Cyclic Peptides 2025 by Iris Biotech GmbH

CYCLIC PEPTIDES

Empowering Peptide Innovation

With this guiding theme in mind, Iris Biotech’s mission is to support researchers by supplying

• innovative technologies,

• rare compounds,

• as well as a broad portfolio on standard consumables, available in flexible quantities from small scale to bulk quantities. To fulfill our dedication “Empowering Peptide Innovation”, we are attending various conferences, symposia, and exhibitions each year. This allows us to remain in direct contact with scientists all over the world, both from academia and industry, to exchange knowledge, and to gather new ideas to tackle your current challenges.

Guided by our dedication to provide

• competent service,

• as well as novel substances and latest technologies,

Iris Biotech is your trusted partner for the world of peptides, while having strong expertise in associated disciplines. Thus, our portfolio comprises reagents and tools for the synthesis and modification of peptides, e.g., amino acids, resins and solvents but also for related technologies such as drug delivery, linkerology® and life sciences.

Owed to the growing demand for tailor-made compounds, our portfolio is fine-tuned by our custom synthesis service at Iris Biotech Laboratories. Our skilled scientists offer profound expertise in de novo route development,

• upscaling towards larger scale production,

• as well as synthesis optimization for increased efficiency.

Examples are the synthesis of rare chiral building blocks, unnatural amino acid derivatives, sophisticated orthogonal protecting groups, heterocycles, building blocks for nucleotides, PEGs and PEG-analogs as well as specific linkers for controlled drug delivery and release.

Portfolio Overview

Peptide Synthesis and Modification

(Protected) Amino Acids

Standards such as Fmoc-D/L-AAA and Boc-D/L-AAA, Smoc amino acids for peptide synthesis in water, variety of protecting groups (e.g., Pbf, Trt, tBu, Bzl, Acm, Mob, SIT, Phacm, Allocam, Mmt), unusual amino acids, fluorinated derivatives, substituted prolines, arginine analogs

Building Blocks

Amino alcohols, amino aldehydes, diamines and hydrazines, (pseudoproline) dipeptides, polyamines and spermines, fatty acid derivatives, peptide nucleic acids (PNAs)

Reagents

Coupling reagents, solvents and scavengers, protecting groups

Resins

Preloaded resins (e.g., based on Trityl, TCP, TentaGel, Methoxybenzhydryl, Merrifield, PAM, Rink, Wang), scavenger resins, hydrazone resins, poly(acrylamide) resins, Cyclover

Linkerology® and Drug Delivery Life Sciences

Linkers for Solid Phase Peptide Synthesis

Cleavable Linkers

Val-Ala-based, Val-Cit-based, disulfide-based, Dde-helping hands, pH-sensitive linkers

Photo-Activatable Linkers

Functionalized Linkers

Clickable linkers, trifunctional linkers, linkers with maleimide function, cross-linkers, selective N-term acylation and biotinylation, 5HP2O

PROTACs

Ligands, linkers & modules

Fullerenes, Poly(2-oxazolines), Dextrans & Plant-Derived Cholesterol

Superparamagnetic Iron Oxide Nanoparticles

Poly-Amino Acids

Poly-Arg, Poly-Glu, Poly-Lys, Poly-Orn, Poly-Sar

PEGylation

Branched PEGylating reagents, (amino-)PEG-acids, PEG-amines & hydrazides & guanidines, reagents for Click-conjugation, Biotin-PEG-reagents, PEG-thiols, PEG-maleimides, other PEGylating reagents

Biotinylation Reagents

Carbohydrates

Galactose, Glucose, Mannose, Xylose and others

Drug Metabolites

Peptides

Substrates & Inhibitors

E.g., protein kinase inhibitors, substrates for fusion (Halo/ Snap/Clip)-tagged proteins

Natural Products

Dyes and Fluorescent Labels

E.g., ICG, AMC, DAPI

Maillard & Amadori Reaction Products

Large portfolio of derivatives useful as standards for food, pharma and cosmetics industry

Vitamins

Custom Synthesis

Your project requires a compound not listed in our portfolio? Get in contact and inquire about our custom synthesis capabilities.

Our experienced scientists are excited to accept your synthetic challenge! In such cases, your request undergoes the following stages:

Step-by-Step Analysis

Customer’s demands

Process Evaluation

Det ailed literature review

Synthetic possibilities

Strategy Development

Protocol development

Method development and validation

• Customized synthesis

Our Service Promise

Quality Consistency

• Identity confirmation Purity verification

All our services are based on high standards, transparency & documentation, trust, honesty & confidentiality, as well as the required know-how.

High Standards

Values: sustainability & responsibility

State-of-the-art equipment & latest technologies

High quality standards

Qualified suppliers & regular audits

Trust, Honesty & Confidentiality

Intergenerational business valuing partnerships

• Meeting the customer‘s expectations

• Integrity towards our customers

Transparency & Documentation

Talk to our specialists – customer care

Certificates of analysis & origin

Impurity profiling

Safety data sheets

• Analytical and process reports

Our Know-How

One-step reactions & complex multi-step synthesis

• Scalability from mg to kg quantities

• Route scouting

2.1. Acid-Labile

2.1.2.

2.1.3.

2.1.4.

2.1.7. 4-Methoxybenzyl (Mob, 4-MBzl)

2.1.8. Methylbenzyl (Meb, 4-MeBn, 4-MeBzl)

2.1.9.

2.4.

2.5.1. Allyloxycarbonyl (Alloc, Aloc)

2.5.2. Allyloxycarbonylaminomethyl (Allocam)

2.5.3. (Allyloxycarbonylamino)phenylacetylaminomethyl (Aapam)

2-Nitroveratryl (oNv)/Dimethoxynitrobenzyl (DMNB)

2.7.2. Nitrodibenzofuran (NDBF)

2.7.3. (Methylenedioxy)nitrophenylethyl (MDNPE)

2.8. Reduction-Labile

(S-Dmp) and

2.8.3. Sec-isoamyl mercaptan (SIT)

2.8.4. 3-Nitro-2-pyridinesulfenyl (Npys)

(S-Tmp)

4.1.5.

4.2.4.

4.2.5.

4.3.

Cyclic Peptides

1. General Introduction

Cyclic peptides have been attracting a lot of attention in recent decades, especially in the area of drug discovery, as more and more naturally occurring cyclic peptides with diverse biological activities, e.g., antibacterial, toxic, immunosuppressive or antitumor activity, have been discovered in all kingdoms of life. Compared to linear peptides, cyclic peptides exhibit a more rigid conformation which is often further increased by additional disulfide bond formation. All together, these bridges allow to form the desired tertiary structure which confers biological activity to the (linear) peptide.

In general, cyclization of peptides results in significantly improved proteolytic stability compared to their linear counterparts. Thus, they are metabolically more stable. Furthermore, the related conformational restriction usually leads to enhanced binding and selectivity as well as increased bioavailability and biological activity as the preorganized ring architecture lowers entropic cost during receptor-binding processes.

Referring to the site of the peptide cyclization reaction, four types can be distinguished: head-to-tail, head-to-side-chain, side-chain-to-tail, and side-chain-to-side-chain cyclization.

Fig. 1: Categorization of peptide cyclization.

Many naturally occurring cyclic peptides are cyclized in the head-to-tail fashion. Within the so-cyclized peptide, the absence of a free N- and C-terminus renders these types of cyclic peptides resistant to hydrolysis by exopeptidases, which further enhances their metabolic stability compared to other types of cyclic peptides.

However, linear precursors without any turn-inducing element typically adopt an extended conformation due to the more stable all-trans configuration of the corresponding amide bonds which positions the N- and C-termini far away from each other, rendering them less likely to react intramolecularly. To prevent intermolecular reactions during in solution cyclization attempts, high dilutions are required that lower overall synthetic efficiency. Performing on-resin cyclization allows to achieve a “pseudo dilution” via immobilization of the linear precursor on the solid support.

References:

→ Ligation Technologies for the Synthesis of Cyclic Peptides; H. Y. Chow, Y. Zhang, E. Mathesn, X. Li; Chem. Rev. 2019; 119(17): 9971-10001. arrow-up-right-from-square https://doi.org/10.1021/acs.chemrev.8b00657

→ Cyclic Peptides as Therapeutic Agents and Biochemical Tools; S. H. Joo; Biomer Ther (Seoul) 2012; 20(1) : 19-26. arrow-up-right-from-square https://doi.org/10.4062/biomolther.2012.20.1.019

→ Cyclic peptides: backbone rigidification and capability of mimicking motifs at protein-protein interfaces; H. Huang, J. Damjanovic, J. Miao, Y.-S. Lin; Phys. Chem. Chem. Phys 2021; 23: 607-616. arrow-up-right-from-square https://doi.org/10.1039/D0CP04633G

→ Cyclic Peptides as Drugs for Intracellular Targets: The Next Frontier in Peptide Therapeutic Development; L. K. Buckton, M. N. Rahimi, S. R. McAlpine; Chem. Eur. J. 2021; 27(5): 1487-1513. arrow-up-right-from-square https://doi.org/10.1002/chem.201905385

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Any Questions or Suggestions?

We are there for you – simply choose one of the numerous possibilities to get in touch!

+49 (0) 9231 97121-0

+49 (0) 9231 97121-99 info@iris-biotech.de www.iris-biotech.de

Cyclic Peptides

2. Cyclization via Disulfide Bond Formation

Formation of intramolecular disulfide bonds by oxidation of the corresponding free thiol precursors is usually the last step in the synthesis of disulfide-containing peptides and is mainly performed in solution. If only a single disulfide bond is to be formed, any available cysteine protecting group compatible with the chosen combination of side-chain and N-α protecting groups can be used.

In contrast, the formation of multiple disulfide bridges in a targeted way to achieve the desired pairing is a significant challenge, in particular if high purity and good yields are required. It already becomes complex, when two disulfide bridges are present, as the four cysteine residues can form three different bridged derivatives: CysI/CysII & CysIII/CysIV, CysI/CysIII & CysII/CysIV, and CysI/CysIV & CysII/CysIII. Therefore, over the last years, increasingly sophisticated strategies for the protection and subsequent deprotection of cysteine have been developed.

In 1977, Barany and Merrifield described the concept of “orthogonality”, which describes protecting groups that can be chemoselectively removed in the presence of one another without affecting/removing each other when applying defined conditions.

In the following, we present a selection of commonly used, as well as innovative side-chain protected cysteines categorized by their cleavage mechanism and available at Iris Biotech. For a detailed review on cysteine protecting groups, please see Org. Process Res. Dev. 2024; 28(1) : 26–45.

References:

→ A new amino protecting group removable by reduction. Chemistry of the dithiasuccinoyl (Dts) function; G. Barany, R. B. Merrifield; J. Am. Chem. Soc. 1977; 99(22): 7363-7365. arrow-up-right-from-square https://doi.org/10.1021/ja00464a050

→ Cysteine protecting groups: applications in peptide and protein science; R. J. Spears, C. McMahon, V. Chudasama; Chem. Soc. Rev. 2021; 50: 11098. arrow-up-right-from-square https://doi.org/10.1039/d1cs00271f

→ Ready to Use Cysteine Thiol Protecting Groups in SPPS, A Practical Overview; A. Chakraborty, S. N. Mthembu, B. G. de la Torre, F. Albericio; Org. Process Res. Dev. 2024; 28(1): 26–45. arrow-up-right-from-square https://doi.org/10.1021/acs.oprd.3c00425

→ Cyclic Peptides for Drug Development; X. Ji, A. L. Nielsen, C. Heinis; Angew. Chem. Int. Ed. 2024; 63(3) : e202308251. arrow-up-right-from-square https://doi.org/10.1002/anie.202308251

→ Native and Engineered Cyclic Disulfide-Rich Peptides as Drug Leads; T. J. Tyler, T. Durek, D. J. Craik; Molecules 2023; 28(7) : 3189. arrow-up-right-from-square https://doi.org/10.3390/molecules28073189

2.1. Acid-Labile Protecting Groups

2.1.1. Trityl (Trt)

For the formation of one disulfide bridge, the common building block Fmoc-Cys(Trt)-OH (FAA1040 on page 79) is used even in bulk productions. It is common to remove Trt using weak acids (e.g., 25% TFA) in the presence of scavengers such as triisopropylsilane (TIS) or triethylsilane (TES) which prevent back-addition of the released Trt cations onto the synthesized peptide during cleavage and isolation. These cleavage conditions render Trt orthogonal to other common protecting groups such as Acm or tBu. Recently, Cys(Trt) has also been shown to be fully deprotected when treated with CuSO 4 and cysteamine in aqueous buffered conditions. The Trt protecting group is compatible with standard Fmoc SPPS reagents.

At Iris Biotech we offer side-chain Trt protected cysteine derivatives either with free N-/C-terminus, in combination with other protecting groups, or as preloaded resins.

Especially, we are also offering the Smoc-L-Cys(Trt)-OH building block (SAA1110 on page 82). The Smoc technology allows to replace organic solvents with water during peptide synthesis and thus represents a greener approach for the production of peptides

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Cyclic Peptides

AAA2015 Aloc-L-Cys(Trt)-OH

N-alpha-Allyloxycarbonyl-S-trityl-L-cysteine

CAS-No. 96865-72-4

Formula C 26 H25NO4 S Mol. weight 447,55 g/mol

BAA1084 Boc-L-Cys(Trt)-OH

N-alpha-t-Butyloxycarbonyl-S-trityl-L-cysteine

CAS-No. 21947-98-8

Formula C 27H29 NO4 S Mol. weight 463,59 g/mol

BAA5000 Boc-D-Cys(Trt)-OH

N-alpha-t-Butyloxycarbonyl-S-trityl-D-cysteine

CAS-No. 87494-13-1

Formula C 27H29 NO4 S Mol. weight 463,59 g/mol

FAA9315

Fmoc-L-Lys(Boc-Cys(Trt))-OH

N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(N-(tert-butoxycarbonyl)-S-trityl-L-cysteinyl)-L-lysine

CAS-No. 587854-43-1

Formula C 48 H 51N3 O 7S Mol. weight 814,01 g/mol

FAA9325 Fmoc-L-Dab(Boc-Cys(Trt))-OH

(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl) amino)-4-((R)-2-((tert-butoxycarbonyl)amino)-3-(tritylthio)propanamido)butanoic acid

CAS-No. 2968514-52-3

Formula C 46 H47N3 O 7S Mol. weight 785,96 g/mol

FAA1035 Fmoc-D-Cys(Trt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-trityl-D-cysteine

CAS-No. 167015-11-4

Formula C 37H31NO4 S Mol. weight 585,71 g/mol

FAA1040 Fmoc-L-Cys(Trt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-trityl-L-cysteine

CAS-No. 103213-32-7

Formula C 37H31NO4 S Mol. weight 585,71 g/mol

FAA1587 Fmoc-L-Pen(Trt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-trityl-L-penicillamine

CAS-No. 201531-88-6

Formula C 39 H35NO4 S Mol. weight 613,78 g/mol

FAA1675 Fmoc-D-Pen(Trt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-trityl-D-penicillamine

CAS-No. 201532-01-6

Formula C 39 H35NO4 S Mol. weight 613,78 g/mol

FAA3570 Fmoc-L-MeCys(Trt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-N-alpha-methyl-S-trityl-L-cysteine

CAS-No. 944797-51-7

Formula C 38 H33 NO4 S Mol. weight 599,74 g/mol

FAA5670 Fmoc-L-Cys(Trt)-OMe

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-trityl-L-cysteine methyl ester

CAS-No. 245088-56-6

Formula C 38 H33 NO4 S Mol. weight 599,74 g/mol

HAA1560 H-L-Cys(Trt)-NH2

S-Trityl-L-cysteine amide

CAS-No. 166737-85-5

Formula C 22H22N2OS Mol. weight 362,49 g/mol

Cyclic Peptides

HAA1995 H-L-Cys(Trt)-Ot Bu*HCl

CAS-No. 158009-03-1

S-Trityl-L-cysteine t-butyl ester hydrochloride S O (R O H2N

Formula C 26 H29 NO 2 S*HCl Mol. weight 419,58*36,45 g/mol

HAA2100 H-D-Cys(Trt)-Ot Bu*HCl

CAS-No. 439089-10-8

S-Trityl-D-cysteine t-butyl ester hydrochloride S O (S) O H2N

Formula C 26 H29 NO 2 S*HCl Mol. weight 419,58*36,45 g/mol

HAA2810 N3 -L-Cys(Trt)-OH*CHA

(R)-2-azido-3-(tritylthio)propanoic acid cyclohexylamine S

CAS-No. 1286670-90-3

Formula C 22H19 N3 O 2 S*C 6 H13 N Mol. weight 389,47*99,17 g/mol

HAA3520 H-D-Cys(Trt)-OMe*HCl

CAS-No. 1020369-32-7

S-trityl-D-cysteine methyl ester hydrochloride S OMe (S) O H2N

Formula C 23 H23 NO 2 S*HCl Mol. weight 377,50*36,45 g/mol

HAA6120 H-D-Cys(Trt)-OH

CAS-No. 25840-82-8

S-Trityl-D-cysteine S OH (S) O H2N

Formula C 22H21NO 2 S Mol. weight 363,48 g/mol

HAA6160 H-L-Cys(Trt)-OH

CAS-No. 2799-07-7

S-Trityl-L-cysteine S OH (R) O H2N

Formula C 22H21NO 2 S Mol. weight 363,48 g/mol

IAD1040 Boc-L-Ser[Fmoc-L-Cys(Trt)]-OH

O-(N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-trityl-L-cysteinyl)-N-(tert-butoxycarbonyl)-L-serine

CAS-No. 944283-13-0

Formula C 45H44N2O 8 S

Mol. weight 772,9

IAD2040 Boc-L-Thr[Fmoc-L-Cys(Trt)]-OH

O-(N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-trityl-L-cysteinyl)-N-(tert-butoxycarbonyl)-L-threonine

CAS-No. 944283-30-1

Formula C 46 H46 N2O 8 S Mol. weight 786,93 g/mol

PYV1140 Fmoc-L-Cys(Trt)-NHN=Pyv Resin

Fmoc-S-trityl-L-cysteinyl-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size

100-200 mesh

Loading > 0.3 mmol/g DVB 1% DVB

RAA1060 H-D-Cys(Trt)-2CT Resin

H-D-Cys(Trt)-2-chlorotrityl resin

Mesh Size

100-200 mesh

Loading > 0.4 mmol/g

DVB 1% DVB

RAA1065 H-L-Cys(Trt)-2CT Resin

H-L-Cys(Trt)-2-chlorotrityl resin

Mesh Size

100-200 mesh

Loading 0.4-1.2 mmol/g

1% DVB

RAA1066 H-L-Cys(Trt)-2CT Resin

H-L-Cys(Trt)-2-chlorotrityl resin

Mesh Size

200-400 mesh

Loading 0.4-1.2 mmol/g

DVB 1% DVB

Cyclic Peptides

SAD1106

Fmoc-D-Cys(Trt)-AC TG

Fmoc-D-Cys(Trt)-[3-methoxy-4-hydroxymethyl)phenoxyacetylamid] TentaGel S

Loading 0.16-0.26 mmol/g

SAD1206 Fmoc-D-Cys(Trt)-Trt TG

Fmoc-D-Cys(Trt)-Trityl TentaGel S

Mesh Size 90 µm

Loading 0.15-0.25 mmol/g

SAD1306 Fmoc-D-Cys(Trt)-Wang TG

Fmoc-D-Cys(Trt)-Wang TentaGel S

Mesh

90 µm

Loading 0.16-0.26 mmol/g

SAL1106 Fmoc-L-Cys(Trt)-AC TG

Fmoc-L-Cys(Trt)-[3-methoxy-4-hydroxymethyl)phenoxyacetylamid] TentaGel S

Mesh Size 90 µm

Loading 0.16-0.26 mmol/g

SAL1206

Fmoc-L-Cys(Trt)-Trt TG

Fmoc-L-Cys(Trt)-Trityl TentaGel S

Mesh Size 90 µm

Loading 0.15-0.25 mmol/g

SAL1306 Fmoc-L-Cys(Trt)-Wang TG

Fmoc-L-Cys(Trt)-Wang TentaGel S

Mesh Size 90 µm

Loading 0.16-0.26 mmol/g

TCP1130 Fmoc-L-Cys(Trt)-TCP-Resin

Fmoc-Cys(Trt)-trityl-carboxyamidomethyl polystyrene

Mesh Size 200-400 mesh

Loading 0.3-0.8 mmol/g DVB 1% DVB

WAA41306 Fmoc-L-Cys(Trt)-Wang Resin

Fmoc-L-Cysteine(Trityl)-Wang Resin

Mesh Size 200-400 mesh DVB 1% DVB

WAA6118 Fmoc-D-Cys(Trt)-Wang Resin

Fmoc-D-Cysteine(Trityl)-Wang Resin

Mesh Size 100-200 mesh DVB 1% DVB

ZAA1310 Z-L-Cys(Trt)-OH

N-alpha-Benzyloxycarbonyl-S-trityl-L-cysteine

CAS-No. 26311-04-6

Formula C 30 H27NO4 S

Mol. weight 497,60 g/mol

SAA1110 Smoc-L-Cys(Trt)-OH

N-(((2,7-disulfo-9H-fluoren-9-yl)methoxy)carbonyl)-S-trityl-L-cysteine potassium salt

CAS-No. 2442552-68-1

Formula C 37H29 K 2NO 10 S 3

Mol. weight 822,01 g/mol

Cyclic Peptides

2.1.2. 4-Methoxytrityl (Mmt)

Mmt represents a very acid-labile cysteine protecting group, which can already be cleaved using 1-3% TFA in DCM/TES. The Mmt group displays orthogonality to multiple cysteine protecting groups, including tBu, Dpm, oNv, StBu, and Acm. It is stable to bases, e.g., 30% piperidine in DMF (24 h, 22 °C), and compatible with standard Fmoc SPPS reagents.

For the targeted synthesis of two disulfide bridges, a commonly applied protecting group combination is Trt and Mmt. As Mmt can be removed with 1% TFA and Trt requires a higher concentration for removal (ca. 7-10%), in principle, a certain degree of orthoganality is given. However, in large scale syntheses it is necessary to utilize 5% TFA for Mmt removal to achieve complete cleavage. At this concentration, it can be observed that a significant percentage of Trt is already cleaved as well. Therefore, no true orthogonality is provided by this protecting group pair. In such cases, it is best to use the protection pair Mmt and Dpm.

FAA1030 Fmoc-L-Cys(Mmt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-p-methoxytrityl-L-cysteine

CAS-No. 177582-21-7

Formula C 38 H33 NO 5 S Mol.

615,74 g/mol

FAA1614 Fmoc-D-Cys(Mmt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-p-methoxytrityl-D-cysteine

CAS-No. 1198791-73-9

Formula C 38 H33 NO 5 S Mol. weight 615,74 g/mol

Product details

Fig. 2: Orthogonality between Mmt and Trt (left), and Mmt and Dpm (right).

HAA3500 H-D-Cys(Mmt)-OH

S-p-methoxytrityl-D-cysteine

CAS-No. 926935-33-3

Formula C 23 H23 NO 3 S

Mol. weight 393,5 g/mol

RAA1055 H-L-Cys(Mmt)-2CT Resin

H-L-Cys(Mmt)-2-chlorotrityl resin

Mesh Size 100-200 mesh

Loading > 0.4 mmol/g DVB 1% DVB

RAA2620 Fmoc-L-Cys(Mmt resin)-NH2

Fmoc-L-Cysteine alpha-amide-S-(4-methoxytrityl resin)

Mesh Size 100-200 mesh

Loading ca. 0.5 mmol/g

DVB 1% DVB

Reference:

→ Synthesis of the very acid-sensitive Fmoc-Cys(Mmt)-OH and its application in solid-phase peptide synthesis; K. Barlos, D. Gatos, O. Hatzi, N. Koch, S. Koutsogianni; Int. J. Pept. Protein Res. 1996; 47: 148-153. arrow-up-right-from-square https://doi.org/10.1111/j.1399-3011.1996.tb01338.x

2.1.3. Diphenylmethyl (Dpm, Bzh, Bh)

Dpm is stable to low concentrations of TFA cocktails (< 25%) but can be cleaved with higher concentrations. At least 60% TFA up to 90% TFA in DCM (using 2.5% TIS and 2.5% H2 O as scavengers) is required for full removal. Due to its acid lability profile, Dpm is orthogonal to Trt and Mmt. Additionally, cysteine racemization is attenuated considerably when using Dpm compared to Trt or Bzl. Furthermore, Dpm is stable towards standard Fmoc SPPS reagents.

Cyclic Peptides

FAA3190 Fmoc-L-Cys(Dpm)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-diphenylmethyl-L-cysteine

CAS-No. 247595-29-5

Formula C 31H27NO4 S

Mol. weight 509,62 g/mol

FAA5650 Fmoc-D-Cys(Dpm)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-diphenylmethyl-D-cysteine

Formula C 31H27NO4 S

Mol. weight 509,62 g/mol

Reference:

→ Acid-labile Cys-protecting groups for the Fmoc/tBu strategy: filling the gap; M. Gongora-Benitez, L. Mendive-Tapia, I. Ramos-Tomillero, A. C. Breman, J. Tulla-Puche, F. Albericio; Org Lett 2012; 14: 54725. arrow-up-right-from-square https://doi.org/10.1021/ol3025

2.1.4. Tetrahydropyranyl (Thp)

Tetrahydropyranyl (Thp) is an S,O -acetal nonaromatic protecting group for cysteine which has been shown to be superior to Trt, Dpm, Acm, and StBu in solid-phase peptide synthesis using the Fmoc/tBu strategy. Thp is stable in mildly acidic conditions (1% TFA in DCM), but its acid-lability is strongly increased in the presence of TIS. Cys(Thp)-protected peptides exist as a diastereomeric mixture; however, once cleaved in concentrated TFA with a scavenger (e.g., 95% TFA, 2.5% TIS in DCM), a single pure product is obtained. Racemization is also decreased compared to Trt, Dpm or StBu, and fewer side products, e.g., C-terminal 3-(1-piperidinyl)alanine adducts, are observed. More recently, Thp has been explored as a protecting group for additional amino acid residues such as serine and threonine.

Furthermore, Thp protection leads to improved solubility of the respective cysteine-containing protected peptides. Complete Thp deprotection can be carried out with either TFA/TIS/DCM (10:1.5:88.5) within 5 min or with HCl/dioxane (12:88) in 2 h. Additionally, Thp is stable to standard Fmoc SPPS reagents.

FAA4160 Fmoc-L-Cys(Thp)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-tetrahydropyranyl-L-cysteine

CAS-No. 1673576-83-4

Formula C 23 H25NO 5 S

Mol. weight 427,15 g/mol

References:

→ Tetrahydropyranyl, a nonaromatic acid-labile Cysteineprotecting group for Fmoc peptide chemistry; I. RamosTomillero, H. Rodriguez, F. Albericio; Org Lett 2015; 17: 1680-3. arrow-up-right-from-square https://doi.org/10.1021/acs.orglett.5b00444

→ Studies on the Synthesis of Insulin Peptides; G. F. Holland, L. A. Cohen; J. Am. Chem. Soc. 1958; 80(14): 3765-3769. arrow-up-right-from-square https://doi.org/10.1021/ja01547a075

→ Understanding Tetrahydropyranyl as a Protecting Group in Peptide Chemistry; A. Sharma, I. Ramos-Tomillero, A. El-Faham, E. Nicolas, H. Rodriguez, B. G. de la Torre, F. Albericio; ChemistryOpen 2017; 6: 168-177. arrow-up-right-from-square https://dx.doi.org/10.1002/open.201600156

2.1.5. tert- Butyl (t Bu)

Cleavage of tBu can be achieved with HF using anisole as scavenger and TFA in the presence of 2,2‘-dithiobis(5-nitropyridine) (DTNP). To form the disulfide, cleavage may be performed using silyl choride-sulfoxide in TFA, Tl(TFA)3 or DMSO in TFA (with DMSO acting as an oxidant). If orthogonality to Meb is desired, DMSO/TFA may also be used for deprotection – tBu is cleaved in DMSO/TFA at room temperature, whilst higher temperatures (45 °C) are required to cleave Meb. The above-mentioned methods of deprotection are rather harsh and often result in the formation of side products and low yields. PdCl 2 in a 50 mM Tris or urea buffer at 37 °C was shown to cleave tBu, providing a much milder way to remove the protecting group. tBu is not removed by [Pd(allyl)Cl]2 making it orthogonal to Thz and Acm under those conditions. Besides this, the Cys(tBu) protecting group is stable to oxidation by I2 , stable towards AgOTf/TFA, neat TFA treatment as well as standard Fmoc SPPS reagents.

Product details

BAA1082 Boc-L-Cys(t Bu)-OH

N-alpha-t-Butyloxycarbonyl-S-t-butyl-L-cysteine

CAS-No. 56976-06-8

Formula C12H23 NO4 S Mol. weight 277,37 g/mol

Cyclic Peptides

FAA1716 Fmoc-L-Cys(t Bu)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-t-butyl-L-cysteine

CAS-No. 67436-13-9

Formula C 22H25NO4 S

Mol. weight 399,51 g/mol

HAA6150 H-L-Cys(t Bu)-OH*HCl

S-t-Butyl-L-cysteine hydrochloride

CAS-No. 2481-09-6

Formula C 7H15NO 2 S*HCl

Mol. weight 177,26*36,45 g/mol

TCP1120 Fmoc-L-Cys(t Bu)-TCP-Resin

Fmoc-Cys(tBu)-trityl-carboxyamidomethyl polystyrene

Mesh Size 200-400 mesh

Loading 0.3-0.8 mmol/g DVB 1% DVB

References:

→ Palladium prompted on-demand cysteine chemistry for the synthesis of challenging and uniquely modified proteins; M. Jbara, S. Laps, M. Morgan, G. Kamnesky, G. Mann, C. Wolberger, A. Brik; Nat. Commun. 2018; 9: 3154. arrow-up-right-from-square https://doi.org/10.1038/s41467-018-05628-0

→ Synthesis of Four-Disulfide Insulin Analogs via Sequential Disulfide Bond Formation; F. Wu, J. P. Mayer, V. M. Gelfanov, F. Liu, R. D. DiMarchi; J. Org. Chem. 2017; 82(7): 3506-3512. arrow-up-right-from-square https://doi.org/10.1021/acs.joc.6b03078 → 2,2‘-Dithiobis(5-nitropyridine) (DTNP) as an effective and gentle deprotectant for common cysteine protecting groups; A. L. Schroll, R. J. Hondal, S. Flemer Jr.; J. Pept. Sci. 2012; 18(1) : 1-9. arrow-up-right-from-square https://doi.org/10.1002/psc.1403

→ Tert-Butyl group as thiol protection in peptide synthesis; J. J. Pastuszak, A. Chimiak; J. Org. Chem. 1981; 46(9) : 1868-1873. arrow-up-right-from-square https://doi.org/10.1021/jo00322a024

2.1.6 Benzyl (Bzl)

The Bzl group is the most stable acid-labile protecting group. The use of S-Bzl protection for cysteine is suitable for Boc/Bzl-based SPPS. The protecting group can be removed using Na/NH 3 or anhydrous HF in the presence of scavengers at room temperature. Nowadays, it is mainly replaced by more labile benzylbased protecting groups such as Mob or Meb.

HAA1574 H-L-Cys(Bzl)-OH

S-Benzyl-L-cysteine

CAS-No. 3054-01-1

Formula C10 H13 NO 2 S Mol. weight 211,29 g/mol

HAA6110 H-D-Cys(Bzl)-OH

S-Benzyl-D-cysteine

CAS-No. 23032-53-3

Formula C10 H13 NO 2 S Mol. weight 211,29 g/mol

HAA6080 H-L-Cys(Bzl)-OMe*HCl

S-Benzyl-L-cysteine methyl ester hydrochloride

CAS-No. 16741-80-3

Formula C11H15NO 2 S*HCl Mol. weight 225,31*36,45 g/mol

BAA1079 Boc-L-Cys(Bzl)-OH

N-alpha-t-Butyloxycarbonyl-S-benzyl-L-cysteine

CAS-No. 5068-28-0

Formula C15H21NO4 S Mol. weight 311,38 g/mol

BAA5410 Boc-D-Cys(Bzl)-OH

N-alpha-t-Butyloxycarbonyl-S-benzyl-D-cysteine

CAS-No. 102830-49-9

Formula C15H21NO4 S Mol. weight 311,38 g/mol

Cyclic Peptides

2.1.7. 4-Methoxybenzyl (Mob, 4-MBzl)

The conditions typically required for full removal of Mob are harsh: neat TFA at 100 °C or HF are reported for deprotection. Mob can also be removed by heavy metal salts, e.g., Hg(TFA)2 or AgOTf in TFA/ thio-anisole, followed by DTT to obtain the free thiol. Tl(TFA)3 can also be used for deprotection. Besides this, deprotection via treatment with PdCl 2 in 50 mM Tris or urea buffer at 37 °C is reported. AgOTf cannot cleave Meb, making Mob and Meb orthogonal under this treatment. The Mob protecting group is stable to HBr, TFA (without scavengers) and standard Fmoc SPPS reagents.

Product details

BAA1081 Boc-L-Cys(Mob)-OH

N-alpha-t-Butyloxycarbonyl-S-(4-methoxy-benzyl)-L-cysteine

CAS-No. 18942-46-6

Formula C16 H23 NO 5 S Mol. weight 341,43 g/mol

FAA1715 Fmoc-L-Cys(Mob)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(4-methoxybenzyl)-L-cysteine

CAS-No. 141892-41-3

Formula C 26 H25NO 5 S Mol. weight 463,55 g/mol

HAA6100 H-L-Cys(Mob)-OH

S-(4-Methoxybenzyl)-L-cysteine

CAS-No. 2544-31-2

Formula C11H15NO 3 S Mol. weight 241,3 g/mol

References:

→ Silver trifluoromethanesulphonate as an S-deprotecting reagent for the synthesis of cystine peptides; N. Fujii, A. Otaka, T. Watanabe, A. Okamachi, H. Tamamura, H. Yajima, Y. Inagaki, M. Nomizu, K. Asano; J. Chem. Soc., Chem. Commun. 1989; 283-284. arrow-up-right-from-square https://doi.org/10.1039/C39890000283

→ Reduction of cysteine-S-protecting groups by triisopropylsilane; E. J. S. Marie, R. J. Hondal; J. Pept. Sci. 2018; 24(11) : e3130. arrow-up-right-from-square https://doi.org/10.1002/psc.3130

→ A New Method for the Protection of the Sulfhydryl Group during Peptide Synthesis; A. Shiro, S. Shumpei, S. Yasutsugu, N. Yoshifumi; Bull. Chem. Soc. Japan 1964; 37(3) : 433-434. arrow-up-right-from-square https://doi.org/10.1246/bcsj.37.433

→ Use of Anhydrous Hydrogen Fluoride in Peptide Synthesis. I. Behavior of Various Protective Groups in Anhydrous Hydrogen Fluoride; S. Shumpei. S. Yasutsugu, K. Yasuo, O. Masanori, S. Hideo; Bull. Chem. Soc. Japan 1967; 40(9): 2164-2167. arrow-up-right-from-square https://doi.org/10.1246/bcsj.40.2164

2.1.8. Methylbenzyl (Meb, 4-MeBn, 4-MeBzl)

The Meb protecting group is broadly similar to Mob but less labile to TFA. Meb can be removed using HF-anisole (50%, 1 h, 0 °C), Tl(TFA)3 or DMSO/TFA (45 °C). Meb is stable towards AgOTf and orthogonal to Trt, Acm, tBu and StBu. Consequently, these protecting groups have frequently been used together. Furthermore, Meb is stable towards standard Fmoc and Boc SPPS reagents.

BAA1080 Boc-L-Cys(MBzl)-OH

N-alpha-t-Butyloxycarbonyl-S-(4-methyl-benzyl)-L-cysteine

CAS-No. 61925-77-7

Formula C16 H23 NO4 S Mol. weight 325,43 g/mol

BAA5420 Boc-D-Cys(MBzl)-OH

N-alpha-t-Butyloxycarbonyl-S-(4-methyl-benzyl)-D-cysteine

CAS-No. 61925-78-8

Formula C16 H23 NO4 S Mol. weight 325,43 g/mol

HAA6090 H-L-Cys(MBzl)-OH

S-(4-Methylbenzyl)-L-cysteine

CAS-No. 42294-52-0

Formula C11H15NO 2 S Mol. weight 225,3 g/mol

FAA1714 Fmoc-L-Cys(MBzl)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(4-methylbenzyl)-L-cysteine

CAS-No. 136050-67-4

Formula C 26 H25NO4 S Mol. weight 447,53 g/mol

References:

Cyclic Peptides

→ Acid stability of several benzylic protecting groups used in solid-phase peptide synthesis. Rearrangement of O-benzyltyrosine to 3-benzyltyrosine; B. W. Erickson, R. B. Merrifield; J. Am. Chem. Soc. 1973; 95(11) : 3750-3756. arrow-up-right-from-square https://doi.org/10.1021/ja00792a046

→ Regioselective Formation of Multiple Disulfide Bonds with the Aid of Postsynthetic S-Tritylation; M. Mochizuki, S. Tsuda, K. Tanimura, Y. Nishiuchi; Org. Lett. 2015; 17(9): 2202-2205. arrow-up-right-from-square https://doi.org/10.1021/acs.orglett.5b00786

2.1.9. 4,4’-Dimethoxydiphenylmethyl (Ddm)

This protecting group can be removed using 10% TFA (TFA:DCM:TIS:H2 O 10:85:2.5:2.5, 1 h, 25 °C). It is recommended as a racemization-suppressing alternative to Trt. Additionally, Ddm can be used as alternative to Mmt, if sterical hindrance of the bulky Mmt group is an issue. Ddm is compatible with standard Fmoc SPPS reagents.

Product details

FAA6940 Fmoc-L-Cys(Ddm)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-di(4-methoxyphenyl)methyl-L-cysteine

CAS-No. 1403825-56-8

Formula C 33 H31NO 6 S Mol. weight 569,67 g/mol

Reference:

→ Evaluation of acid-labile S-protecting groups to prevent cysteine racemization in Fmoc solid-phase peptide synthesis; H. Hibino, Y. Miki, Y. Nishiuchi; J. Pept. Sci. 2014; 20(1) : 30-35. arrow-up-right-from-square https://doi.org/10.1002/psc.2585

2.1.10. Pseudoprolines (ψPro)

Pseudoprolines have developed as standard building blocks for peptide synthesis in order to disrupt aggregation, reduce aspartimide formation and thus help improving the yield of difficult and long sequences. Cysteine-based pseudoprolines are usually more stable than the corresponding oxazolidines of serine and threonine.

The proline-resembling thiazolidine ring of Cys-pseudoprolines simultaneously protects the side-chains and amino group of cysteine. Typically, pseudoprolines are incorporated into peptides using dipeptide building blocks. In the case of ψMe,MePro, the protected cysteine can be deprotected by TFA within hours.

This can be reduced to minutes when using ψH,DmpPro. Complete Cys(ψMe,MePro) deprotection can alternatively be achieved with TFMSA at 0 °C within 15 min. Furthermore, ψH,DmpPro shows stability to Pd(0) suggesting orthogonality to allyl-based protecting groups. Alternatively, for ψH,HPro, the group is stable to strong acids. Besides this, in general, the kinetics of deprotection appear to be dependent on the acid and solvent system used. Notably, the use of pseudoprolines induces a “kink” within the peptide backbone – similar to proline – which favors peptide macrocyclization.

PSI1440 Fmoc-Gly-L-Cys[PSI(Dmp,H)pro]-OH

(S)-3-(N-(9-Fluorenylmethyloxycarbonyl)-glycyl)-2-(2,4-dimethoxphenyl)thiazolidine-4-carboxylic acid

CAS-No. 1926163-05-4

Formula C 29 H28 N2O 7S Mol. weight 548,61 g/mol

PSI1450 Fmoc-L-Ala-L-Cys[PSI(Dmp,H)pro]-OH

(S)-3-(N-(9-Fluorenylmethyloxycarbonyl)-L-alanyl)-2-(2,4-dimethoxphenyl)thiazolidine-4-carboxylic acid

CAS-No. 2022956-37-0

Formula C 30 H30 N2O 7S Mol. weight 562,63 g/mol

PSI1470 Fmoc-L-Asp(t Bu)-L-Cys[PSI(Dmp,H)pro]-OH

(S)-3-(N-(9-Fluorenylmethyloxycarbonyl)-beta-t-butyl-L-aspartyl)-2-(2,4-dimethoxphenyl)thiazolidine-4-carboxylic acid

CAS-No. 1359754-16-7

Formula C 35H39 N2O 9 S Mol. weight 662,75 g/mol

PSI1490 Fmoc-L-Glu(t Bu)-L-Cys[PSI(Dmp,H)pro]-OH

(S)-3-(N-(9-Fluorenylmethyloxycarbonyl)-gamma-t-butyl-L-glutamyl)-2-(2,4-dimethoxphenyl)thiazolidine-4-carboxylic acid

CAS-No. 2565804-44-4

Formula C 36 H40 N2O 9 S Mol. weight 676,78 g/mol

PSI1510 Fmoc-L-Leu-L-Cys[PSI(Dmp,H)pro]-OH

(S)-3-(N-(9-Fluorenylmethyloxycarbonyl)-L-leucyl)-2-(2,4-dimethoxphenyl)thiazolidine-4-carboxylic acid

CAS-No. 1926163-06-5

Formula C 33 H36 N2O 7S Mol. weight 604,71 g/mol

Cyclic Peptides

PSI1520 Fmoc-L-Lys(Boc)-L-Cys[PSI(Dmp,H)pro]-OH

(S)-3-(N-(9-Fluorenylmethyloxycarbonyl)-N-epsilon-t-butyloxycarbonyl-L-lysyl)-2-(2,4-dimethoxphenyl) thiazolidine-4-carboxylic acid

CAS-No. 1926163-07-6

Formula C 38 H45N3 O 9 S Mol. weight 719,84 g/mol

PSI1560 Fmoc-L-Tyr(t Bu)-L-Cys[PSI(Dmp,H)pro]-OH

(S)-3-(N-(9-Fluorenylmethyloxycarbonyl)-O-t-butyl-L-thyrosyl)-2-(2,4-dimethoxphenyl)thiazolidine-4-carboxylic acid

Formula C 40 H42N2O 8 S Mol. weight 710,84 g/mol

PSI1570 Fmoc-L-Val-L-Cys[PSI(Dmp,H)pro]-OH

(S)-3-(N-(9-Fluorenylmethyloxycarbonyl)-L-valyl)-2-(2,4-dimethoxphenyl)thiazolidine-4-carboxylic acid

CAS-No. 1926163-08-7

Formula C 32H34N2O 7S Mol. weight 590,69 g/mol

PSI1580 Fmoc-L-Cys(Trt)-L-Cys(Psi(Dmp,H)pro)-OH

(R)-3-(N-(9-Fluorenylmethyloxycarbonyl)-S-trityl-L-cysteinyl)-2-(2,4-dimethoxphenyl)thiazolidine-4-carboxylic acid

CAS-No. 2022956-75-6

Formula C 49 H44N2O 7S 2 Mol. weight 837,01 g/mol

References:

→ Pseudo-Prolines as a Molecular Hinge: Reversible Induction of cis Amide Bonds into Peptide Backbones; P. Dumy, M. Keller, D. E. Ryan, B. Rohwedder, T. Wöhr, M. Mutter; J. Am. Chem. Soc. 1997; 119: 918-925. arrow-up-right-from-square https://doi.org/10.1021/ja962780a

→ Pseudo-Prolines as a Solubilizing, Structure-Disrupting Protection Technique in Peptide Synthesis; T. Wöhr, F. Wahl, A. Nefzi, B. Rohwedder, T. Sato, X. Sun, M. Mutter; J. Am. Chem. Soc. 1996; 118: 9218-9227. arrow-up-right-from-square https://doi.org/10.1021/ja961509q

→ Expediting the Fmoc solid phase synthesis of long peptides through the application of dimethyloxazolidine dipeptides; P. White, J. W. Keyte, K. Bailey and G. Bloomberg; J. Pept. Sci. 2004; 10: 18-26. arrow-up-right-from-square https://doi.org/10.1002/psc.484

→ Incorporation of Pseudoproline Derivatives Allows the Facile Synthesis of Human IAPP, a Highly Amyloidogenic and Aggregation-Prone Polypeptide; A. Abedini, D. P. Raleigh; Org. Lett. 2005; 7(4): 693-696. arrow-up-right-from-square https://doi.org/10.1021/ol047480+

→ An improved synthetic and purification procedure for the hydrophobic segment of the transmembrane peptide phospholamban; E. K. Tiburu, P. C. Dave, J. F. Vanlerberghe, T. B. Cardon, R. E. Minto, G. A. Lorigan; Anal. Biochem. 2003; 138(1): 146-151. arrow-up-right-from-square https://doi.org/10.1016/S0003-2697(03)00141-6

→ Synthesis of Cyclogossine B Using a Traceless Pseudoproline Turn-Inducer; M. S. Y. Wong, K. A. Jilliffe; Aust. J. Chem. 2009; 63(5) : 797-801. arrow-up-right-from-square https://doi.org/10.1071/CH09643

2.2. Oxidation-Labile Protecting Group Acetamidomethyl (Acm)

Acm shows stability to commonly used peptide synthesis protocols, can be removed under relatively mild conditions and displays no major racemization problems. The Acm protecting group works orthogonal to a number of other cysteine protecting groups, e.g., Trt, tBu, Meb, Msbh, Mmt, and Dnpe. One deprotection method, for example, is the removal by using transition metal catalysts, such as Pd(II) complexes. Besides, cleavage can be triggered by using 15 eq. DTNP in 97.5% TFA/thioanisole, I2 , or Hg(OAc)2

One major disadvantage of the Acm group is the tendency of the cleaved protecting group to alkylate the electron rich aromatic rings of tyrosine and tryptophan. The synthesis of the following model sequence (including tyrosine and tryptophane) with Fmoc-Cys(Acm)-OH as building block clearly shows the three expected impurities in significant concentrations in the case of using Acm.

Target sequence:

Ala – Cys– Phe – Trp – Lys – Tyr – Cys– Val

Side products:

Ala – Cys– Phe – Trp(Acm) – Lys – Tyr – Cys– Val

Ala – Cys– Phe – Trp – Lys – Tyr(Acm) – Cys– Val

Ala – Cys– Phe – Trp(Acm) – Lys – Tyr(Acm) – Cys– Val

Fig. 3: Side products formed by back-alkylation when using Acm as cysteine protecting group.

Alternatives to the Acm protecting group are represented by Phacm and Allocam.

BAA1078 Boc-L-Cys(Acm)-OH

N-alpha-t-Butyloxycarbonyl-S-(acetyl-aminomethyl)-L-cysteine

CAS-No. 19746-37-3

Formula C11H20 N2O 5 S

Mol. weight 292,36 g/mol

FAA1506 Fmoc-L-Cys(Acm)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(acetyl-aminomethyl)-L-cysteine

CAS-No. 86060-81-3

Formula C 21H22N2O 5 S

Mol. weight 414,48 g/mol

Cyclic Peptides

FAA6230 Fmoc-D-Cys(Acm)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(acetyl-aminomethyl)-D-cysteine

CAS-No. 168300-88-7

Formula C 21H22N2O 5 S

Mol. weight 414,48 g/mol

HAA6070 H-L-Cys(Acm)-OH*HCl

S-(Acetyl-aminomethyl)-L-cysteine hydrochloride

CAS-No. 28798-28-9

Formula C 6 H12N2O 3 S*HCl

Mol. weight 192,24*36,45 g/mol

SAD1107 Fmoc-D-Cys(Acm)-AC TG

Fmoc-D-Cys(S-Acm)-[3-methoxy-4-hydroxymethyl) phenoxyacetylamid] TentaGel S

Mesh Size 90 µm

Loading 0.16-0.26 mmol/g

SAD1207 Fmoc-D-Cys(Acm)-Trt TG

Fmoc-D-Cys(S-Acm)-Trityl TentaGel S

Mesh Size 90 µm

Loading 0.15-0.25 mmol/g

SAD1307 Fmoc-D-Cys(Acm)-Wang TG

Fmoc-D-Cys(S-Acm)-Wang TentaGel S

Mesh Size 90 µm

Loading 0.16-0.26 mmol/g

SAL1107 Fmoc-L-Cys(Acm)-AC TG

Fmoc-L-Cys(S-Acm)-[3-methoxy-4-hydroxymethyl) phenoxyacetylamid] TentaGel S

Mesh Size 90 µm

Loading 0.16-0.26 mmol/g

SAL1207

Fmoc-L-Cys(Acm)-Trt TG

Fmoc-L-Cys(S-Acm)-Trityl TentaGel S

Mesh Size 90 µm

Loading 0.15-0.25 mmol/g

SAL1307 Fmoc-L-Cys(Acm)-Wang TG

Fmoc-L-Cys(S-Acm)-Wang TentaGel S

Mesh Size 90 µm

Loading 0.16-0.26 mmol/g

TCP1110

Fmoc-L-Cys(Acm)-TCP-Resin

Fmoc-Cys(Acm)-trityl-carboxyamidomethyl polystyrene

Mesh Size 200-400 mesh

Loading 0.3-0.8 mmol/g

DVB 1% DVB

WAA11307 Fmoc-L-Cys(Acm)-Wang Resin

Fmoc-L-Cys(S-Acm)-Wang Resin

Mesh Size 100-200 mesh DVB 1% DVB

WAA41307 Fmoc-L-Cys(Acm)-Wang Resin

Fmoc-L-Cys(S-Acm)-Wang Resin

Mesh Size 200-400 mesh

DVB 1% DVB

References:

→ Palladium-Mediated Direct Disulfide Bond Formation in Proteins Containing S-Acetamidomethyl-cysteine under Aqueous Conditions; S. Laps, H. Sun, G. Kamnesky, A. Brik; Angew. Chem. Int. Ed. 2019; 58(17) : 5729-5733. arrow-up-right-from-square https://doi.org/10.1002/anie.201900988

→ Studies on deprotection of cysteine and selenocysteine side-chain protecting groups; K. M. Harris, S. Flemer Jr, R. J. Hondal; J. Pept. Sci. 2007; 13(2) : 81-93. arrow-up-right-from-square https://doi.org/10.1002/psc.795

→ p-Nitrobenzyl protection for cysteine and selenocysteine: A more stable alternative to the acetamidomethyl group; M. Muttenthaler, Y. Garcia Ramos, D. Feytens, A. D. de Araujo, P. F. Alewood; Pept. Sci. 2010; 94(4): 423-432. arrow-up-right-from-square https://doi.org/10.1002/bip.21502

Cyclic Peptides

2.3. Base-Labile Protecting Groups

Base-labile protecting groups include 9-fluorenylmethyl (Fm; cleavage with eg. HF:anisole 95:5, 1 h, 0 °C), 2-(2,4-dinitrophenyl)ethyl (Dnpe; cleavage using 50% piperidine in DMF, 30 min), 9-Fluorenylmethoxy-carbonyl (Fmoc; transformation to Fm by treatment with Et 3 N). Like all other base-labile protecting groups, they are incompatible with Fmoc SPPS.

2.4. Enzyme-Labile Protecting Groups

2.4.1.

Phenylacetamidomethyl (Phacm)

Phacm shows the same stability and orthogonality as Acm and has the additional advantage that it can be deprotected either chemically by I2/AcOH, or enzymatically by Penicillin G Amidase (PGA). Phacm is stable to standard Fmoc and Boc SPPS reagents.

Through the mild and highly specific conditions when removing Phacm with PGA, no adduct formation occurs and the desired cyclic peptide is being formed in high yield. This also holds true if Phacm is removed chemically with I2 . PGA (penicillin G amidase, penicillin acylase, penicillin amidohydrolase from E. coli on acrylic resin, systematic name: penicillin amidohydrolase, E.C. 3.5.1.11) has an active pocket which is very specific for phenylacetic acid (Phac). The most prominent commercial use is hydrolysis of a phenylacetamid bond during production of the penicillin API 6-APA (DeMartin et al., J. Mol. Catal. B: Enzymatic 1999; 6: 437). The high specifity of PGA towards the phenylacetyl moiety makes the use of Phacm very promising as alternative for Acm. The principle capability of using PGA for hydrolyzing Phacm and deprotecting cysteine already was discovered by Albericio et al. in 1995 using native PGA. Adding co-solvents like acetonitrile helps to improve the solubility of hydrophobic sequences. Water in combination with DMSO (80:20) will make deprotection and cyclization a one-pot reaction, which is usually completed within 16 h to 24 h (37 °C, pH 7).

Peptide Synthesis with Fmoc-Cys(Acm)

Adduct Formation with Trp and Tyr:

Several impurities present in significant concentration of peptides carrying Tyr(Acm), Trp(Acm).

Orthogonality: Fmoc, Boc

Deprotection: TFA, Npys, I2 , Tl 3+, Ph2 SO, MeSiCl 3

Peptide Synthesis with Fmoc-Cys(Phacm)

No Adduct Formation:

• clean crude peptide

• clean product and high yield

Orthogonality: same as Acm

Deprotection: same as Acm and by hydrolysis in the presence of PGA, water:DMSO (80:20), pH 7, 37 °C, 16-24 h

Fig. 4: Comparison of Fmoc-Cys(Acm) and Fmoc-Cys(Phacm) as building blocks for peptide synthesis.

Isolated in 1997 off the coast of Mozambique from marine species, the cyclothiodepsipeptide thiocoraline has been identified as a potent antitumor agent. Four N-methylated amino acids and two of them in D configuration mask a DNA bisintercalating chromophore. It has several features that make its structure extremely complex. In particular the high number of cysteines, the presence of consecutive N-methylamino acids, and a bicyclic structure formed by a disulfide bridge flanked by two thioester moieties, makes it a real challenge for synthetic chemists.

Albericio et al. mastered the synthesis by selecting an orchestrated scheme of protecting groups where the key technology was using the phenylacetamidomethyl (Phacm) group, since it can be cleaved under very mild, i.e. aqueous, conditions.

Cyclic Peptides

Fig. 5: Chemical structure of the cyclothiodepsipeptide thiocoraline.

BAA6390 Boc-L-Cys(Phacm)-OH

N-alpha-t-Butyloxycarbonyl-S-(Phenylacetylaminomethyl)-L-cysteine

CAS-No. 57084-73-8

Formula C17H24N2O 5 S Mol. weight 368,45 g/mol

FAA3710 Fmoc-D-Cys(Phacm)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(phenylacetylaminomethyl)-D-cysteine

CAS-No. 1565818-55-4

Formula C 27H26 N2O 5 S Mol. weight 490,57 g/mol

FAA6910 Fmoc-L-Cys(Phacm)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-Phenylacetylaminomethyl)-L-cysteine

CAS-No. 159680-21-4

Formula C 27H26 N2O 5 S Mol. weight 490,57 g/mol

Product details

References:

→ Enzyme-labile protecting groups for the synthesis of natural products: solid-phase synthesis of thiocoraline; J. Tulla-Puche, M. Gongora-Benitez, N. Bayo-Puxan, A. M. Francesch, C. Cuevas, F. Albericio; Angew. Chem. Int. Ed. Engl. 2013; 52: 5726-30. arrow-up-right-from-square https://doi.org/10.1002/anie.201301708

→ S-Phenylacetamidomethyl (Phacm): an orthogonal cysteine protecting group for Boc and Fmoc solid-phase peptide synthesis strategies; M. Royo, J. Alsina, E. Giralt, U. Slomcyznska, F. Albericio; J. Chem. Soc., Perkin Trans. 1 1995; 1095-1102. arrow-up-right-from-square https://doi.org/10.1039/p19950001095

2.5.

Palladium-Labile Protecting Groups

2.5.1. Allyloxycarbonyl (Alloc, Aloc)

Alloc groups can be deprotected using tributyltin hydride (Bu 3 SnH) and a Pd(0) catalyst. Removal can be achieved within 10 min using PdCl 2 (PPh 3)2 in DCM and AcOH with Bu 3 SnH. The Alloc group is stable in TFA/DCM (24 h, 50 °C), but base-labile. Piperidine treatment (30% in DMF, 3 h, 30 °C) of Boc-Cys(Alloc)–OH results in complete removal of the Alloc group. Additionally, under Fmoc conditions, the Alloc group is prone to undergoing β arrow-right α shifts, and intramolecular acylation reactions may also occur. These issues hinder its suitability as a cysteine protecting group. As such, other allyl-based Pd-labile protecting groups, such as Allocam, are recommended instead.

References:

→ Allyl-based groups for side-chain protection of amino-acids; A. Loffet, H. X. Zhang; Int. J. Pept. Prot. Res. 1993; 42(4): 346-351. arrow-up-right-from-square https://doi.org/10.1111/j.1399-3011.1993.tb00504.x

→ Use of Alloc-amino acids in solid-phase peptide synthesis. Tandem deprotection-coupling reactions using neutral conditions; N. Thieriet, J. Alsina, E. Giralt, F. Guibé, F. Albericio; Tetrahedron Lett 1997; 38: 7275-7278. arrow-up-right-from-square https://doi.org/10.1016/s0040-4039(97)01690-0

2.5.2. Allyloxycarbonylaminomethyl (Allocam)

The Allocam-protecting group, which is a variant of the Acm protecting group, allows for a palladium-mediated single-step approach using mild reaction conditions and readily available reagents. The Allocam group can be removed using Bu 3 SnH and a Pd(0) catalyst in AcOH (10 min, RT). Cys(Allocam) displays stability towards piperidine but is slightly unstable to the acidic conditions used for Boc removal, with ~10% degradation seen following 20 h of treatment with 25% TFA in DCM. Besides this, Allocam is stable to standard Fmoc SPPS reagents.

Cyclic Peptides

FAA7610 Fmoc-L-Cys(Allocam)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-((allyloxycarbonylamino)methy)-L-cysteine

CAS-No. 232953-09-2

Formula C 23 H24N2O 6 S

Mol. weight 456,51 g/mol

References:

→ Direct palladium-mediated on-resin disulfide formation from Allocam protected peptides; T. D. Kondasinghe, H. Y. Saraha, S. B. Odeesho, J. L. Stockdill; Org Biomol Chem 2017; 15: 2914-2918. arrow-up-right-from-square https://doi.org/10.1039/c7ob00536a

→ Disulfide Formation Strategies in Peptide Synthesis; T. M. Postma, F. Albericio; Eur. J. Org. Chem. 2014; 17: 35193530. arrow-up-right-from-square https://doi.org/10.1002/ejoc.201402149

→ Allylic protection of thiols and cysteine: I: The allyloxycarbonylaminomethyl group; A. Malanda Kimbonguila, A. Merzouk, F. Guibé, A. Loffet; Tetrahedron 1999; 55: 6931-6944. arrow-up-right-from-square https://doi.org/10.1016/s0040-4020(99)00322-1

→ Allylic protecting groups and their use in a complex environment part II: Allylic protecting groups and their removal through catalytic palladium π-allyl methodology; F. Guibé; Tetrahedron 1998; 54: 2967-3042. arrow-up-right-from-square https://doi.org/10.1016/s0040-4020(97)10383-0

→ The allyloxycarbonylaminomethyl group: a new allytic protection for the thiol group of cysteine; A. M. Kimbonguila, A. Merzouk, F. Guibé, A. Loffet; Tetrahedron Lett 1994; 35: 9035-9038. arrow-up-right-from-square https://doi.org/10.1016/0040-4039(94)88420-x

2.5.3. (Allyloxycarbonylamino)phenylacetylaminomethyl (Aapam)

Cys(Aapam) represents an Alloc protected Phacm linker as removable side-chain modification for the incorporation of e.g., solubility tags facilitating the preparation of hydrophobic peptides and proteins. The Alloc-Phacm cysteine can easily be introduced in peptides during Fmoc SPPS. The Alloc group can then be removed by using tetrakis(triphenylphosphine)palladium(0) [Pd(PPh 3) 4] in the presence of phenylsilane with the Phacm group being completely stable under these conditions. After Alloc-deprotection, further groups can be coupled to the remaining free amine of Phacm, e.g., a solubility tag. The fully synthesized peptide can be cleaved from the resin by using TFA and the solubilized peptide fragments can be assembled by ligation. Finally, the Phacm-linked solubility tag can easily be removed in solution by treatment with PdCl 2 to yield the fully unprotected cysteine side-chain. As the resulting free cysteine can be converted to alanine through desulfurization, the Alloc-Phacm linker can be used as side-chain modification to incorporate solubility tag at the position of cysteine, but also of alanine.

FAA5150 Fmoc-L-Cys(Aapam)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-((4-(allyloxycarbonylamino)phenylacetylaminomethyl)-L-cysteine

CAS-No. 1946783-89-6

Formula C 31H31N3 O 7S Mol. weight 589,66 g/mol

Product details

References:

→ Palladium Mediated Rapid Deprotection of N-Terminal Cysteine under Native Chemical Ligation Conditions for the Efficient Preparation of Synthetically Challenging Proteins; M. Jbara, S. K. Maity, M. Seenaiah, A. Brik; J. Am. Chem. Soc. 2016; 138(15) : 5069-5075. arrow-up-right-from-square https://doi.org/10.1021/jacs.5b13580

→ Palladium-assisted removal of a solubilizing tag from a Cysteine Side Chain to Facilitate Peptide and Protein Synthesis; S. K. Maity, G. Mann, M Jbara, S. Laps, G. Kamnesky, A. Brik; Org. Lett. 2016; 18(12) : 3026-3029. arrow-up-right-from-square https://doi.org/10.1021/acs.orglett.6b01442

2.6. N-terminal Cysteine Protecting Groups

2.6.1. Thiazolidine (Thz)

The Thz group simultaneously protects the thiol and the amino group of cysteine and can be removed using oxidants such as H2 O 2 and I2 . Removal will also occur following treatment with iodoacetic acid and benzyl chloride (pH 10–11, RT), or ferric chloride in air (pH 10). Alternatively, deprotection of Thz can be achieved by adjusting the reaction mixture to ca. pH 4 in the presence of a large excess of methoxyamine.

Product details

BAA1135 Boc-L-Thz-OH

(R)-N-t-Butyloxycarbonyl-thiazolidine-4-carboxylic acid

CAS-No. 51077-16-8

Formula C9 H15NO4 S Mol. weight 233,29 g/mol

BAA1186 Boc-D-Thz-OH

(S)-N-(t-Butyloxycarbonyl)-thiazolidine-4-carboxylic acid

CAS-No. 63091-82-7

Formula C9 H15NO4 S Mol. weight 233,29 g/mol

FAA1427 Fmoc-L-Thz-OH

(R)-N-(9-Fluorenylmethyloxycarbonyl)-thiazolidine-L-4-carboxylic acid

CAS-No. 133054-21-4

Formula C19 H17NO4 S Mol. weight 355,42 g/mol

Cyclic Peptides

FAA1437 Fmoc-L-Thz(Me2)-OH

(R)-N-(9-Fluorenylmethyloxycarbonyl)-2,2-dimethyl-thiazolidine-4-carboxylic acid

CAS-No. 873842-06-9

Formula C 21H21NO4 S Mol. weight 383,46 g/mol

FAA1495 Fmoc-D-Thz-OH

(S)-N-alpha-(9-Fluorenylmethyloxycarbonyl)-thiazolidine-4-carboxylic acid

CAS-No. 198545-89-0

Formula C19 H17NO4 S Mol. weight 355,42 g/mol

HAA1132 H-L-Thz-OH

(R)-Thiazolidine-4-carboxylic acid

CAS-No. 34592-47-7

Formula C 4H7NO 2 S Mol. weight 133,16 g/mol

FAA9320 Fmoc-L-Lys(Boc-Thz)-OH

N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-((R)-3-(tert-butoxycarbonyl)thiazolidine-4-carbonyl)-L-lysine

Formula C 30 H37N3 O 7S Mol. weight 583,70 g/mol

FAA9330 Fmoc-L-Dab(Boc-Thz)-OH

(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl) amino)-4-((R)-3-(tert-butoxycarbonyl)thiazolidine-4-carboxamido)butanoic acid

CAS-No. 2968514-54-5

Formula C 28 H33 N3 O 7S Mol. weight 555,65 g/mol

References:

→ The Action of Formaldehyde upon Cysteine; S. Ratner, H. T. Clarke; J. Am. Chem. Soc. 1937; 59(1): 200-206. arrow-up-right-from-square https://doi.org/10.1021/ja01280a050

→ Palladium in the Chemical Synthesis and Modification of Proteins; M. Jbara, S. Kumar Maity, A. Brik; Angew. Chem. Int. Ed. 2017; 56(36) : 10644-10655. arrow-up-right-from-square https://doi.org/10.1002/anie.201702370

2.7. Photolabile Protecting Groups

2.7.1. 2-Nitroveratryl (o Nv)/Dimethoxynitrobenzyl (DMNB)

2-Nitroveratryl (oNv) is a photolabile orthogonal protecting group that is compatible with SPPS protocols and can be cleaved by irradiation with UV light (350 nm, 30 min, aq. media) under ambient conditions. No significant racemization is observed upon incorporation of Cys(oNv) during SPPS using diisopropyl-carbodiimide (DIC) and HOBt activation. Combination with S-pyridinesulfenyl activation allows for rapid in situ disulfide bond formation. In order to demonstrate the versatility of this approach, it was applied to the synthesis of a number of model peptides, e.g., oxytocin, α-conotoxin ImI, and human insulin.

FAA3970 Fmoc-L-Cys(o Nv)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(2-nitroveratryl)-L-cysteine

CAS-No. 214633-71-3

Formula C 27H26 N2O 8 S Mol. weight 538,57 g/mol

FAA8870 Fmoc-L-hCys(o Nv)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(2-nitroveratryl)-L-homocysteine

Formula C 28 H28 N2O 8 S Mol. weight 552,60 g/mol

HAA9320 H-L-Cys(o Nv)-OH

S-(4,5-dimethoxy-2-nitrobenzyl)-L-cysteine

CAS-No. 214633-68-8

Formula C12H16 N2O 6 S Mol. weight 316,33 g/mol

Reference:

→ 2-nitroveratryl as a photocleavable thiol-protecting group for directed disulfide bond formation in the chemical synthesis of insulin; J. A. Karas, D. B. Scanlon, B. E. Forbes, I. Vetter, R. J. Lewis, J. Gardiner, F. Separovic, J. D. Wade, M. A. Hossain; Chemistry 2014; 20: 9549-52. arrow-up-right-from-square https://doi.org/10.1002/chem.201403574

→ A method for directed evolution and functional cloning of enzymes; H. Pedersen, S. Hölder, D. P. Sutherlin, U. Schwitter, D. S. King, P. G. Schultz; PNAS 1998; 95(18) : 10523-10528. arrow-up-right-from-square https://doi.org/10.1073/pnas.95.18.10523

→ Light Activation of Protein Splicing with a Photocaged Fast Intein; W. Ren, A. Ji. H.-W. Ai; J. Am. Chem. Soc. 2015; 137(6): 2155-2158. arrow-up-right-from-square https://doi.org/10.1021/ja508597d

Cyclic Peptides

→ Light-Activation of DNA-Methyltransferases; J. Wolffgramm, B. Buchmuller, S. Palei, A. Muñoz-López, J. Kanne, P. Janning, M. R. Schweiger, D. Summerer; Angew. Chem. Int. Ed. 2021; 60(24): 13507-13512. arrow-up-right-from-square https://doi.org/10.1002/anie.202103945

→ Biosynthetic selenoproteins with genetically-encoded photocaged selenocysteines; R. Rakauskaitė, G. Urbanavičiūtė, A. Rukšėnaitė, Z. Liutkevičiūtė, R. Juškėnas, V. Masevičius, S. Klimašauskas; Chem. Commun. 2015; 51: 8245-8248. arrow-up-right-from-square https://doi.org/10.1039/C4CC07910H

2.7.2. Nitrodibenzofuran (NDBF)

Nitrodibenzofuran (NDBF) is a photocleavable side-chain protecting group that can be removed by photolysis upon irradiation with UV-light (365 nm) or – especially for in vivo applications – by two-photon excitation using near infrared light (800 nm). Additionally, NDBF deprotection results in clean conversion to the free thiol without the occurrence of S-to-N shifts. In addition, this cage exhibits a faster UV photolysis rate relative to simple nitroveratryl derivatives. An example showed that NDBF is photolyzed 16–160 times more efficiently than other nitrobenzyl PPGs. Besides, NDBF is fully compatible with Fmoc SPPS. Additional methoxy-substitution (OMe-NDBF) leads to a higher two-photon photolysis efficiency compared to NDBF. Product details

FAA8420 Fmoc-L-Cys(NDBF)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(1-(3-nitro-dibenzofuran-2-yl)-ethyl)-L-cysteine

CAS-No.

1895883-28-9

Formula C 32H26 N2O 7S

Mol. weight 582,62 g/mol

References:

→ A red shifted two-photon-only caging group for three-dimensional photorelease; Y. Becker, E. Unger, M. A. H. Fichte, D. A. Gacek, A. Dreuw, J. Wachtveitl, P. J. Walla, A. Heckel; Chem. Sci. 2018; 9: 2797-2902. arrow-up-right-from-square https://doi.org/10.1039/c7sc05182d

→ Nitrodibenzofuran: A One- and Two-Photon Sensitive Protecting Group That Is Superior to Brominated Hydroxycoumarin for Thiol Caging in Peptides; M. M. Mahmoodi, D. Abate-Pella, T. J. Pundsack, C. C. Palsuledesai, P. C. Goff, D. A. Blank, M. D. Distefano; J. Am. Chem. Soc. 2016; 138: 5848-5859. arrow-up-right-from-square https://doi.org/10.1021/jacs.5b11759

→ The nitrodibenzofuran chromophore: a new caging group for ulta-efficient photolysis in living cells; A. Momotake, N. Lindegger, E. Niggli, R. J. Barsotti, G. C. R. Ellis-Davies; Nature Methods 2006; 3: 35-40. arrow-up-right-from-square https://doi.org/10.1038/NMETH821

→ Methoxy-Substituted Nitrodibenzofuran-Based Protecting Group with an Improved Two-Photon Action Cross-Section for Thiol Protection in Solid Phase Peptide Synthesis; T. K. Bader, F. Xu, M. H. Hodny, D. A. Blank, M. D. Distefano; J. Org. Chem. 2020; 85: 1614-1625. arrow-up-right-from-square https://doi.org/10.1021/acs.joc.9b02751

2.7.3. (Methylenedioxy)nitrophenylethyl (MDNPE)

Cys(MDNPE) can be uncaged by irradiation with 365 nm leading to the formation of a ketone as byproduct, which will not undergo undesired reactions with proteins.

HAA9270 H-L-Cys(MDNPE)-OH

1-[4‘,5‘-(methylenedioxy)-2‘-nitrophenyl]ethyl]-L-cysteine

CAS-No. 1551078-43-3

Formula C12H14N2O 6 S Mol. weight 314,31 g/mol

FAA7945 Fmoc-L-Cys(MDNPE)-OH

N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl)-L-cysteine

Formula C 27H24N2O 8 S Mol. weight 536,56 g/mol

References:

→ Genetic Encoding of Photocaged Cysteine Allows Photoactivation of TEV Protease in Live Mammalian Cells; D. P. Nguyen, M. Mahesh, S. J. Elsässer, S. M. Hancock, C. Uttamapinant, J. W. Chin; J. Am. Chem. Soc. 2014; 136(6): 2240-2243. arrow-up-right-from-square https://doi.org/10.1021/ja412191m

→ Spatio-Temporal Photoactivation of Cytotoxic Proteins; R. Cruz-Samperio, R. J. Mart, L. Y. P. Luk, Y.-H. Tsai, A. T. Jones, R. K. Allemann; ChemBioChem 2022; 23: e202200115. arrow-up-right-from-square https://doi.org/10.1002/cbic.202200115

2.8. Reduction-Labile Protecting Groups

2.8.1. tert- Butylthio

(St Bu)

StBu can be removed under organic or aqueous conditions with reducing agents such as thiols, e.g., 2-mercaptoethanol or DTT, or phosphines (e.g., PBu 3 , PPh 3 , TCEP). The StBu protecting group is stable to acidic conditions (e.g., TFA:thioanisole:phenol, 92:2.5:2.5 v/v) provided no thiol scavenger is added. If required, 2-methylindole and anisole can be used as alternative scavengers. The StBu protecting group is orthogonal to other protecting groups, such as Trt, Acm, Meb/Mob, tBu, and Allocam. StBu is unstable towards HF and can be applied for both Fmoc and Boc SPPS.

Cyclic Peptides

BAA6415 Boc-L-Cys(St Bu)-OH

N-(tert-butoxycarbonyl)-S-(tert-butylthio)-L-cysteine

CAS-No. 30044-61-2

Formula C12H23 NO4 S 2 Mol. weight 309,44 g/mol

FAA1575 Fmoc-L-Cys(St Bu)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(t-butylthio)-L-cysteine

CAS-No. 73724-43-3

Formula C 22H25NO4 S 2 Mol. weight 431,57 g/mol

FAA1965 Fmoc-D-Cys(St Bu)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(t-butylthio)-D-cysteine

CAS-No. 501326-55-2

Formula C 22H25NO4 S 2 Mol. weight 431,57 g/mol

FAA3340 Fmoc-L-MeCys(S-t Bu)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-N-alpha-methyl-S-(t-butylthio)-L-cysteine

CAS-No. 1013096-03-1

Formula C 23 H27NO4 S 2 Mol. weight 445,59 g/mol

HAA6140 H-L-Cys(St Bu)-OH

S-Thio-t-butyl-L-cysteine

CAS-No. 30044-51-0

Formula C 7H15NO 2 S 2 Mol. weight 209,32 g/mol

SAD1109 Fmoc-D-Cys(SS-t Bu)-AC TG

Fmoc-D-Cys(S-S-tBu)-[3-methoxy-4-hydroxymethyl) phenoxyacetylamid] TentaGel S

Mesh Size 90 µm

Loading 0.16-0.26 mmol/g

SAD1209

Fmoc-D-Cys(SS-t Bu)-Trt TG

Fmoc-D-Cys(S-S-tBu)-Trityl TentaGel S

Mesh Size 90 µm

Loading 0.15-0.25 mmol/g

SAD1309

Fmoc-D-Cys(SS-t Bu)-Wang TG

Fmoc-D-Cys(S-S-tBu)-Wang TentaGel S

Mesh Size 90 µm

Loading 0.16-0.26 mmol/g

SAL1109

Fmoc-L-Cys(SS-t Bu)-AC TG

Fmoc-L-Cys(S-S-tBu)-[3-methoxy-4-hydroxymethyl) phenoxyacetylamid] TentaGel S

Mesh Size 90 µm

Loading 0.16-0.26 mmol/g

SAL1209

Fmoc-L-Cys(SS-t Bu)-Trt TG

Fmoc-L-Cys(S-S-tBu)-Trityl TentaGel S

Mesh Size 90 µm

Loading 0.15-0.25 mmol/g

SAL1309

Fmoc-L-Cys(SS-t Bu)-Wang TG

Fmoc-L-Cys(S-S-tBu)-Wang TentaGel S

Mesh Size 90 µm

Loading 0.16-0.26 mmol/g

TCP1230 Fmoc-L-Cys(St Bu)-TCP-Resin

Fmoc-Cys(StBu)-trityl-carboxyamidomethyl polystyrene

Mesh Size

200-400 mesh

Loading 0.3-0.8 mmol/g DVB 1% DVB

Cyclic Peptides

2.8.2.

WAA11309 Fmoc-L-Cys(SS-t Bu)-Wang Resin

Fmoc-L-Cys(S-S-tBu)-Wang Resin

O O S S Mesh Size 100-200 mesh DVB 1% DVB

O (R) O

WAA41309 Fmoc-L-Cys(SS-t Bu)-Wang Resin

Fmoc-L-Cys(S-S-tBu)-Wang Resin

O S S Mesh Size 200-400 mesh DVB 1% DVB

O (R)

References:

→ Trimethoxyphenylthio as a Highly Labile Replacement for tert-Butylthio Cysteine Protection in Fmoc Solid Phase Synthesis; T. M. Postma, M. Giraud, F. Albericio; Org. Lett. 2012; 14(21): 5468-5471. arrow-up-right-from-square https://doi.org/10.1021/ol3025499

→ S-Alkylmercapto-Gruppen zum Schutz der SH Funktion des Cysteins; I. Synthese und Stabilität einiger S-(Alkylmercapto)cysteine; U. Weber, P. Hartter; Hoppe-Seyler’s Z. Physiol. Chem. 1970; 351: 1384-1388. arrow-up-right-from-square https://doi.org/10.1515/bchm2.1970.351.2.1384

Dimethoxyphenylthio (S-Dmp) and

2,4,6-Trimethoxyphenylthio

(S-Tmp)

Both groups can be removed using NMM (0.1 M) with either 20% 2-mercaptoethanol in DMF or 5% DTT in DMF in 5 min. Due to the fast deprotection with nucleophiles, S-Dmp can even be used in automated synthesizers. In contrast, removal of the StBu protecting group using the aforementioned conditions required 3 h of incubation with BME, whereas little to no deprotection of StBu was observed when using DTT. Both the S-Dmp and S-Tmp groups were noted to be compatible to Fmoc removal conditions (20% piperidine in DMF, 4 h).

FAA3180 Fmoc-L-Cys(S-DMP)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(2,6-dimethoxythiophenol)-L-cysteine

CAS-No. 1403834-73-0

Formula C 26 H25NO 6 S 2

Mol. weight 511,61 g/mol

Reference:

2.8.3. Sec-isoamyl mercaptan (SIT)

The new thiol protecting group sec-isoamyl mercaptan (SIT) expands the toolbox for the synthesis of peptides containing multiple disulfide bridges. The building block Fmoc-L-Cys(SIT)-OH (FAA8495 on page 77) is fully compatible with Fmoc solid phase peptide synthesis (SPPS), highly stable towards piperidine (basic conditions) and labile towards disulfide reducing agents. The secondary thiol SIT is more stable than primary ones but easier to remove than tertiary thiols such as StBu.

In a comparative study (Chakraborty et al., 2020), the deprotection rate of Fmoc-Cys(StBu)-OH and FmocCys(SIT)-OH by using DTT as reducing agent was monitored by HPLC for 500 min. After that time, StBu was only partially (60%) removed. In contrast, after already 160 min, SIT was totally removed. Notably, the addition of 5% of water speeds up both reactions: StBu was completely removed within 250 min and SIT in less than 40 minutes. Furthermore, compared to the protecting groups StBu and Trt, SIT shows less racemization.

Product details

FAA8495 Fmoc-L-Cys(SIT)-OH

CAS-No. 2545642-31-5

Formula C 23 H27NO4 S 2

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(sec-isoamyl mercaptan)-L-cysteine (R) H N S OH O O O S

Mol. weight 445,59 g/mol

Cyclic Peptides

FAA8865 Fmoc-L-hCys(SIT)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(sec-isoamyl mercaptan)-L-homocysteine

Formula C 24H29 NO4 S 2 Mol. weight 459,62 g/mol

2.8.4.

References:

→ Disulfide-Based Protecting Groups for the Cysteine Side Chain; A. Chakraborty, A. Sharma, F. Albericio, B. G. de la Torre; Org. Lett. 2020; 22(24): 9644-9647. arrow-up-right-from-square https://doi.org/10.1021/acs.orglett.0c03705

→ olid-Phase Synthesis of an “Inaccessible” hGH-Derived Peptide Using a Pseudoproline Monomer and SITProtection for Cysteine; S. Rao Manne, A. Chakraborty, K. Rustler, T. Bruckdorfer, B. G. de la Torre, F. Albericio; ACS Omega 2022; 7(32) : 28487-28492. arrow-up-right-from-square https://doi.org/10.1021/acsomega.2c03261

3-Nitro-2-pyridinesulfenyl (Npys)

Npys can be removed within 10 min using aliphatic thiols (e.g., 3-mercaptoacetic acid). Besides, deprotection can be triggered at room temperature by tertiary phosphines in the presence of H2 O. Cys(Npys) is stable to strong acids such as TFA (24 h, RT), HF (1h, RT) and 4 M HCl/dioxane, and is thus suitable for Boc SPPS. S-Npys is somewhat stable towards aromatic thiols, which can cleave the O -Npys and N-Npys derivatives, enabling a degree of selectivity. The Npys group is not compatible with Fmoc chemistry. Npys is not stable under basic conditions like piperidine.

Product details

BAA1860 Boc-L-Cys(Npys)-OH

N-alpha-t-Butyloxycarbonyl-S-(3-nitro-2-pyridylthio)-L-cysteine

CAS-No. 76880-29-0

Formula C13 H17N3 O 6 S 2 Mol. weight 375,42 g/mol

HAA3510 H-L-Cys(Npys)-OH*HCl

S-(3-nitro-2-pyridylthio)-L-cysteine hydrochloride

CAS-No. 108807-66-5

Formula C 8 H9 N3 O4 S 2*HCl

Mol. weight 275,30*36,45 g/mol

References:

→ Boc-Cys(Npys)-OH (BCNP): an appropriate reagent for the identification of T cell epitopes in cystine and/or cysteine-containing proteins; G. Mourier, B. Maillèrea, J. Cottona, M. Hervéa, S. Leroya, M. Léonettia, A. Ménez; J. Immunol. Methods 1994; 171(1,2): 65-71. arrow-up-right-from-square https://doi.org/10.1016/0022-1759(94)90229-1

→ Sulfur protection with the 3-nitro-2-pyridinesulfenyl group in solid-phase peptide synthesis; R. J. Ridge, G. R. Matsueda, E. Haber, R. Matsueda; Int. J. Pept. Prot. Res. 1982; 19(5) : 490-498. arrow-up-right-from-square https://doi.org/10.1111/j.1399-3011.1982.tb02634.x

→ 3-Nitro-2-pyridinesulfenyl group: synthesis and applications to peptide chemistry; C. Rentier, K. Fukumoto, A. Taguchi, Y. Hayashi; J. Pept. Sci. 2017; 23(7-8) : 496-504. arrow-up-right-from-square https://doi.org/10.1002/psc.2964

2.9. Safety-Catch Protecting Group

4,4‘-Bis(dimethylsulfinyl)benzhydryl (Msbh)

The term “safety-catch protecting group” means that the protecting group is stable/”safe” to a particular set of conditions until the group undergoes a specific reaction. Msbh is stable to acidic (TFA, HF), oxidative (and reductive conditions) until its electron-withdrawing sulfoxide groups are reduced. The formed sulfide renders the bond between the cysteine sulfhydryl group and the benzylic carbon of the Msbh group acid-labile and thus facilitates deprotection using TFA. Both steps can be performed in a one pot reaction using NH4I/DMS/TFA. The innovative Msbh protecting group is stable to the deprotection conditions of most common cysteine PGs such as Mmt, Trt, Acm, or Phacm. Moreover, it is stable to conditions applied in both Boc and Fmoc chemistry.

TFA/scavenger

carbeniumion target peptide

Fig. 6: Cleavage of the Msbh cysteine protecting group.

Cyclic Peptides

Another suitable application for Msbh-protected cysteine is whenever there is an odd number of cysteines present in a peptide, where all cysteines except one are disulfide-brigded. In such cases, Msbh can be utilized to protect the side-chain of the one cysteine that is supposed to remain a free thiol and is therefore deprotected after all disulfide bonds have been installed. The advantage of using Msbh is that it may greatly reduce the risk of disulfide shuffling during deprotection of the last cysteine residue.

Product details

FAA4155 Fmoc-L-Cys(Msbh)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(4,4‘-dimethylsulfinylbenzhydryl)-L-cysteine

CAS-No. 1584646-97-8

Formula C 33 H31NO 6 S 3

Mol. weight 633,80 g/mol

FAA8150 Fmoc-D-Cys(Msbh)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(4,4‘-dimethylsulfinylbenzhydryl)-D-cysteine

Formula C 33 H31NO 6 S 3

Mol. weight 633,80 g/mol

References:

→ Total synthesis of human hepcidin through regioselective disulfide-bond formation by using the safety-catch cysteine protecting group 4,4’-dimethylsulfinylbenzhydryl; Z. Dekan, M. Mobli, M. W. Pennington, E. Fung, E. Nemeth, P. F. Alewood; Angew. Chem. Int. Ed. Engl. 2014; 53: 2931-4. arrow-up-right-from-square https://doi.org/10.1002/anie.201310103

→ A new safety-catch protecting group and linker for solid-phase synthesis; S. Thennarasu, C.-F. Liu; Tetrahedron Lett. 2010; 51: 3218-3220. arrow-up-right-from-square https://doi.org/10.1016/j.tetlet.2010.04.047

→ A Reductive Acidolysis Final Deprotection Strategy in Solid Phase Peptide Synthesis Based on Safety-Catch Protection; T. Kimura, T. Fukui, S. Tanaka, K. Akaji, Y. Kiso; Chem. Pharm. Bull. 1997; 45: 18-26. arrow-up-right-from-square https://doi.org/10.1248/cpb.45.18

→ A safety-catch type of amide protecting group; M. Pátek, M. Lebl; Tetrahedron Lett. 1990; 31: 5209-5212. arrow-up-right-from-square https://doi.org/10.1016/s0040-4039(00)97844-4

→ The p-(methylsulfinyl)benzyl group: a trifluoroacetic acid (TFA)-stable carboxyl-protecting group readily convertible to a TFA-labile group; J. M. Samanen, E. Brandeis; J. Org. Chem. 1988; 53: 561-569. arrow-up-right-from-square https://doi.org/10.1021/jo00238a016

→ Solid-Phase Peptide Synthesis Using a Four-Dimensional (Safety-Catch) Protecting Group Scheme; S. Noki, E. Brasil, H. Zhang, T. Bruckdorfer, B. G. de la Torre, F. Albericio; J. Org. Chem. 2022, 87(15): 9443–9453. arrow-up-right-from-square https://doi.org/10.1021/acs.joc.2c01056

2.10. Summary of Cysteine Protecting Groups available at Iris Biotech

Tab. 1: Overview of cysteine protecting groups and required removal conditions.

Protecting Group Removal conditions

Acetamidomethyl (Acm) I 2 DTNP

(2,2’-dithiobis (5-nitropyridine)

Tl(III)

Hg(II)

Methyl-o-nitropiperonyl (MDNPE)

Diphenylmethyl (Dpm)

UV-Light (< 365 nm)

Remarks

• Stable to standard peptide synthesis conditions

• Removed under mild conditions with low impact on racemization

Orthogonal to Trt, t Bu, Mbzl, Msbh, and Mmt

Caution must be taken when I 2 is applied for the deprotection as iodination of Trp and Tyr might occur

• Site-specific covalent protein modification possible

60% TFA Used in Fmoc and Boc strategy

• Dpm is orthogonal to Trt and Mmt

Applied as Bzl replacement

Phenyl-acetamidomethyl (Phacm)

I 2/AcOH

4,5-Dimethoxy-2-nitrobenzyl (oNv or DMNB)

4-(Allyloxycarbonylamino) phenylacetylaminomethyl (Aapam)

UV-light (> 350 nm)

Environmentally-friendly alternative to Acm

• Similar stability and lability to Acm

• Deprotection by E. coli penicillin G acylase possible

Orthogonal to Fm, Dnpe, Mbzl Partially orthogonal to Acm

• Stable to Fmoc SPPS conditions Racemization below 0.5%

Pd/Bu 3 SnH/ AcOH

• Used for side-chain modification by solubilizing tags

Cyclic Peptides

Protecting Group Removal conditions

Trityl (Trt) 25% TFA I 2

Remarks

• Standard protecting group in Fmoc strategy

Benzyl (Bn, Bzl) Na/NH 3 HF

TMSBr/TFA/ thioanisol

4-Methylbenzyl (Mbzl) HF, MeSiCl 3 HF/p-cresol

DMSO/TFA (45 min)

Propargyl [(PhCH2NEt 3)2MoS 4]

4-Methoxybenzyl (Mob)

3-Nitro-2-pyridinesulfenyl (Npys)

TFA/TIS Hg(II) Tl(III)

aliphatic thiols (MAA or BME) tertiary phosphines in water

4-Methoxytrityl (Mmt) 2% TFA

Requires harsh deprotection conditions

• Similar to Mob but less susceptible to TFA

Is orthogonal to Trt, Acm, t Bu, and StBu

• Click conjugation

Standard protecting group in Boc strategy

Full removal requires very harsh conditions

Also compatible with Fmoc strategy

• Cys(Mob) might undergo oxidation

Not compatible with Fmoc SPPS but Boc strategy

• Not stable towards TBAF

• Applied in solid phase disulfide ligation

Orthogonal to tBu, Dpm, oNv, StBu, and Acm

Allyloxy-carbonylaminomethyl (Allocam)

Pd(0)/Bu 3 SnH/ AcOH

• Orthogonal to tBu, Dpm, oNv, StBu, and Acm

Structure

Protecting Group

Removal conditions

tert-Butylthio (S-tBu) thiols, phosphines

Dimethoxyphenylthio (S-DMP)

20% BME in DMF 5% DTT in DMF

DABDT, DIPEA/H2 O/ACN (3:3:94)

Nitrodibenzofuran (NDBF)

UV-light (> 365 nm)

Remarks

• Compatible with Boc and Fmoc SPPS

• Orthogonal to Trt, Acm, Mbzl, Mob, and tBu.

More labile alternative for StBu

• Compatible with Fmoc SPPS

Use more TFA-labile resin if selective removal is desired.

Two-photon deprotection is also possible at 800 nm

• Compatible with Fmoc strategy

• Application in living cells possible

4,4-Bis(dimethylsulfinyl) benzhydryl (Msbh)

NH 4I/DMS/TFA

Me 3 SiCl/Ph 3 P

• Safety catch protecting group Stable to Boc and Fmoc strategy

3-(4-Methoxybenzyl)sulfonyl (SO 2 Mob)

Tetrahedropyranyl (THP)

TFMSA/TFA/ H2 O (50:45:5)

• Used to provide cysteine sulfonic acid

2.5% TFA/TIS/ DCM

AgNO 3

tert-Butyl (tBu)

TFMSA, Hg(II)

4,4‘-dimethoxybenzhydryl (Ddm)

TFA/DCM/TIS/ H2 O (10:85:2.5:2.5)

Selective removal in mild acidic conditions

• Better atom economy that Trt Decreased racemization in comparison with Trt, Dpm or StBu

Regioselective deprotection in presence of Trt, Acm and Mmt

Not removed by [Pd(allyl)Cl] 2 therefore in might be used as a orthogonal group to Thz and Acm

Provides lowest suppression rates of racemization in Fmoc SPPS

Cyclic Peptides

Protecting Group Removal conditions

Sec-isoamyl mercaptan 3-methyl-2butanethiol (SIT)

Pseudoproline (thiazolidine)

BME in DMF (1:4), 0.1 M DIPEA 20 eq. DTT, ACN/ DIPEA/H2 O (95:5:5)

95% TFA and scavengers

Remarks

• Stable to Fmoc SPPS conditions

Structure

Thiazolidine (Thz)

H2 O 2 , I 2 Pd(II) and MPAA/TCEP or GSH/ 6 M Gdn · HCl (pH 6.5, 37 °C, 45 min)

Incorporation of a kink into the peptide backbone

• Reduces aggregation during peptide assembly

• In combination with Asp: limits aspartimide formation

Combined protection of thiol and amino group

• Widely used in ligation strategies, but not stable towards NaNO 2 treatment in hydrazide ligation

• Pd-mediated deprotection was reported in vivo to provide an α-oxo aldehyde moiety which can be used for bioconjugation

. circle-arrow-right

For a detailed overview of protecting groups and deprotection conditions, see our brochure „Protecting Groups“!

2.11. Examples

An advanced combination for the targeted synthesis of peptides with more than one disulfide bridge is as follows:

1. Using Mmt as protecting group and removing it with 5% TFA for construction of the first disulfide bridge. By utilizing a 5% TFA solution instead of 1%, the complete deprotection of all Mmt groups can be ensured.

2. Using Dpm with 90% TFA as deprotection condition for the second disulfide bridge. Through this new combination of protecting groups and cleavage conditions, high purity and yield of the desired doublebridged peptide is the result, which makes this combination useful as the solution of choice from research scale through commercial scale productions.

3. A third disulfide bridge can be introduced by using a pair of Phacm-protected cysteines that can be deprotected either chemically or enzymatically.

4. Tetracystine peptides (four disulfide bridges) may be accessed by utilizing a pair of Msbh-protected cysteines. Msbh is a safety-catch protecting group stable to both Boc- and Fmoc-conditions, that only becomes acid-labile after reductive treatment with NH4I/DMS.

90% TFA

I2/AcOH

NH4I/DMS, TFA

Fig. 7: Consecutive disulfide bond formation via use of orthogonal protecting groups.

Cyclic Peptides

Using some of the above-mentioned protecting groups, another potential sequence for the synthesis of a tetracystine peptide is as follows:

1. The first SS bridge can be designed by using S-DMP as cysteine protection, followed by deprotection with DTT.

2. Then Mmt finds its place for building bridge #2. For cleavage, 5% TFA will work well.

3. The third disulfide bond can successfully be put together applying Dpm and deprotecting it with 90% TFA.

4. Bridge #4 is synthesized using Phacm, while mild removal in water will leave all other bridges intact and maintain the effort done so far with building one of the previous bridges after the other.

Numerous other combinations as suggested above can be applied depending on the individual situation and application. The table below demonstrates how the new arsenal of protecting groups can be applied for different numbers of disulfide bridges or other derivatizations.

Tab. 2: Recommended protection pattern for the subsequent formation of several disulfide bridges.

# of Disulfide Bridges Cysteine Building Blocks to Use Deprotection Conditions Oxidative Deprotection

None or 1

Uneven number of Cys (w/wo derivatization)

Fmoc-Cys(Trt)-OH > 25% TFA I2 , Tl 3+

Fmoc-Cys(Mmt)-OH

Fmoc-Cys(Dpm)-OH

Fmoc-Cys(Mmt)-OH

Fmoc-Cys(Dpm)-OH

Fmoc-Cys(Phacm)-OH

Fmoc-Cys(Mmt)-OH

Fmoc-Cys(Dpm)-OH

Fmoc-Cys(Phacm)-OH

Fmoc-Cys(Msbh)-OH

Fmoc-Cys(Trt)-OH

Fmoc-Cys(Msbh)-OH

Or:

Fmoc-Cys(Trt)-OH

Fmoc-Cys(Phacm)-OH

And several other combinations

0.5% to 5% TFA 90% TFA I2 , Tl 3+

0.5% to 5% TFA

90% TFA

I2/AcOH or PGA/water

0.5% to 5% TFA 90% TFA I2/AcOH or PGA/water

NH 4I/TMS, TFA

> 25% TFA

NH4I/TMS, TFA > 25% TFA

Immob. PGA/water

TFA:DMSO:anisole(89:10:1)

I 2 , Tl 3+

TFA:DMSO:anisole(89:10:1)

Immob. PGA/water & DMSO; Ph 2 SO/MeSiCl 3

I 2 , Tl 3+

TFA:DMSO:anisole(89:10:1)

Immob. PGA/water & DMSO; Ph 2 SO/MeSiCl 3

I 2 , Tl 3+

(Immob.) PGA/water & DMSO; Ph 2 SO/MeSiCl 3

3. Cleland’s Reagent – DTT

Cleland’s reagent, also known as DL-Dithiothreitol or DTT, is a water-soluble protective reagent for sulfhydryl groups. It reduces disulfide linkages to free sulfhydryl groups in proteins and enzymes. It is a component of buffers used in protocols for the isolation and purification of proteins.

DTT is a very strong reducing agent due to the property to form a six-membered ring with an internal disulfide bond in oxidized form. The redox potential is -0.33 V at pH 7. The pK a values of the thiol groups are 9.2 and 10.1, respectively. The reduction of a typical disulfide bond proceeds by two sequential thiol-disulfide exchange reactions. The reducing power of DTT is limited to pH values above 7, since only the negatively charged thiolate form is the reactive agent in opening disulfide bonds. DTT is also used as a reducing agent for thiolated DNA. The terminal sulfurs of thiolated DNA have a tendency to oxidize and form dimers in solution, especially in the presence of oxygen. Dimerization significantly lowers the efficiency of subsequent coupling reactions such as DNA immobilization on gold surfaces in biosensors. Normally, DTT is mixed with a DNA solution and allowed to react, and then is removed by filtration (solid catalyst) or by chromatography (liquid form).

DTT is frequently used to reduce the disulfide bonds of proteins and in order to prevent intramolecular (cyclization) and intermolecular (oligomerisation, polymerization) disulfide bonds from cysteine residues of proteins. However, DTT cannot reduce solvent-inaccessible disulfide bonds, so reduction of disulfide bonds is sometimes carried out under denaturing conditions (e.g., at high temperatures, or in the presence of strong denaturating agents such as 6 M guanidinium chloride, 8 M urea, or 1% sodium dodecylsulfate). Conversely, the solvent exposure of different disulfide bonds can be assayed by their speed of reduction in the presence of DTT. DTT can also be used as an oxidizing agent. Its inherent advantage is that effectively no mixed-disulfide species will be formed, which can occur with other agents such as glutathione.

RL-1020 DTT (racemic)

DL-Dithiothreitol

CAS-No. 3483-12-3

Formula C 4H10 O 2 S 2

Mol. weight 154,25 g/mol

References:

→ Dithiothreitol, a New Protective Reagent for Sh Groups; W. W. Cleland; Biochemistry 1964; 3: 480-2. arrow-up-right-from-square https://doi.org/10.1021/bi00892a002

→ Reductive cleavage of cystine disulfides with tributylphosphine; U. T. Rüegg, J. Rudinger; Enzyme Structure Part E C. H. W. Hirs, N. T. Serge 1977; 47: 111-116. arrow-up-right-from-square https://doi.org/10.1016/0076-6879(77)47012-5

→ From production of peptides in milligram amounts for research to multi-tons quantities for drugs of the future; T. Bruckdorfer, O. Marder, F. Albericio; Curr Pharm Biotechnol 2004; 5: 29-43. arrow-up-right-from-square https://doi.org/10.2174/1389201043489620

4. Chemoselective Ligation-Mediated Peptide Cyclization

Within the following chapter, various methods for intramolecular cyclization either creating nonpeptide or peptide linkages are summarized. X Y chemoselective ligation

Fig. 8: Intramolecular cyclization.

The descriptions below should just give an overview about the different technologies available. For a detailed insight on ligation technologies for the synthesis of cyclic peptides, please see the review published by Li et al

Reference:

→ Ligation Technologies for the Synthesis of Cyclic Peptides; H. Y. Chow, Y. Zhang, E. Matheson, X. Li; Chem. Rev. 2019; 119(17): 9971-10001. arrow-up-right-from-square https://doi.org/10.1021/acs.chemrev.8b00657

4.1. Non-Peptide Linkages

4.1.1. Oxime and Hydrazone Ligation

Replacing the primary amino group of an amino acid by an aminooxy moiety leads to an increase in nucleophilicity. Thus, after completion of the peptide synthesis and deprotection of the aminooxy-function, chemoselective reactions with carbonyl compounds (either aldehydes or ketones) under formation of a kinetically stable oxime bond can be performed (= oxime ligation).

Fig. 9: Schematic illustration of oxime ligation.

In the same manner, hydrazine acids, meaning the amino group of an amino acid replaced by a hydrazide moiety, can undergo hydrazone ligation upon reaction with aldehydes or ketones. Hydrazone linkage is reversible but was found to be stable at physiological pH.

Fig. 10: Schematic illustration of hydrazone ligation.

Both oxime and hydrazone linkage represent good peptidomimetics for amide bonds. The application of oxime and hydrazone ligation for the preparation of head-to-tail cyclic peptides relies on the accessibility of the peptide precursors with reactive groups on N- and C-termini, regardless of the absolute position of the two reactive groups. The oxime and hydrazone ligations are highly chemoselective because the reactive moieties (aminooxy, hydrazide, and aldehyde) are biorthogonal to the side-chain functionalities of the 20 natural amino acids. As typical for intramolecular cyclization reactions, high dilution is required to prevent intermolecular ligations.

The low usage of these ligations for head-to-tail cyclic peptide synthesis is most probably due to the possible hydrolysis of the hydrazine and oxime linkage. The formation of a mixture of products with E/Z isomers and the potential side reactions that may occur arising from the instability of the aldehyde or aminooxy-containing precursor cause these ligations to be a less attractive strategy when it comes to head-to-tail cyclic peptide synthesis.

In addition to ligation, another application of hydrazides is their conversion to C-terminal azides with isoamyl nitrite under acidic conditions, and subsequent head-to-tail cyclization forming a native peptide bond.

SPPS

TFA/H2O/TIS (95:2.5:2.5)

Fig. 11: Formation of cyclic peptides via transformation of peptide hydrazides to their azides.

Besides this, peptide hydrazides can also be converted to peptide thioesters, which are intermediates for application in native chemical ligation (NCL), or used directly as thioester surrogates in NCL

Cyclic Peptides

FAA8460 Fmoc-L-cis-Hyp(NHBoc)-OH

Fmoc-4-(Boc-amino)oxy)-proline (2S,4S)

CAS-No. 1015426-31-9

Formula C 25H28 N2O 7 Mol. weight 468,50 g/mol

FAA8450 Fmoc-AAHA(Boc)-OH (S)

(S)-2-(Fmoc-amino)-6-(Boc-aminoxy)hexanoic acid

CAS-No. 357278-11-6

Formula C 26 H32N2O 7 Mol. weight 484,54 g/mol

FAA8445 Fmoc-Hcan(Boc)-OH (S)

(S)-2-(Fmoc-amino)-5-(Boc-aminoxy)pentanoic acid

CAS-No. 204844-15-5

Formula C 25H30 N2O 7 Mol. weight 470,51 g/mol

FAA8455 Fmoc-L-trans-Hyp(NHBoc)-OH

Fmoc-4-(Boc-amino)oxy)-proline (2S,4R)

CAS-No. 1015426-45-5

Formula C 25H28 N2O 7 Mol. weight 468,50 g/mol

Besides this, Iris Biotech is offering hydrazone resins for a reliable and convenient method to synthesize peptide hydrazides. The hydrazone linker is completely stable in the course of standard Fmoc SPPS. Linker cleavage occurs with 95% TFA (e.g., TFA/H2 O/TIS, 95:2.5:2.5), which directly affords the desired peptide as a hydrazide. The hydrazone linker tolerates treatment with 5% TFA/DCM, thus permitting selective removal of Mtt and similar acid-labile protecting groups for on-resin side-chain functionalization.

Product details

PYV1000 Fmoc-NHN=Pyv Resin

Fmoc-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size 100-200 mesh

Loading > 0.3 mmol/g DVB 1% DVB

PYV1100

Fmoc-L-Ala-NHN=Pyv Resin

Fmoc-L-alanyl-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size

100-200 mesh

Loading > 0.3 mmol/g DVB 1% DVB

PYV1110

Fmoc-L-Arg(Pbf)-NHN=Pyv Resin

Fmoc-N‘-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginyl-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size

100-200 mesh

Loading > 0.3 mmol/g DVB 1% DVB

PYV1120

Fmoc-L-Asn(Trt)-NHN=Pyv Resin

Fmoc-N-beta-trityl-L-asparaginyl-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size

100-200 mesh

Loading > 0.3 mmol/g

DVB 1% DVB

PYV1130

Fmoc-L-Asp(Ot Bu)-NHN=Pyv Resin

Fmoc-L-aspartyl-beta-t-butyl ester-alpha-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size

100-200 mesh

Loading > 0.3 mmol/g

DVB 1% DVB

PYV1140

Fmoc-L-Cys(Trt)-NHN=Pyv Resin

Fmoc-S-trityl-L-cysteinyl-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size

100-200 mesh

Loading > 0.3 mmol/g

DVB 1% DVB

PYV1150

Fmoc-L-Glu(t Bu)-NHN=Pyv Resin

Fmoc-L-glutamyl-gamma-t-butyl ester-alpha-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size

100-200 mesh

Loading > 0.3 mmol/g

DVB 1% DVB

Cyclic Peptides

PYV1160

Fmoc-L-Gln(Trt)-NHN=Pyv Resin

Fmoc-N-gamma-trityl-L-glutaminyl-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size

100-200 mesh

Loading > 0.3 mmol/g

DVB 1% DVB

PYV1170

Fmoc-Gly-NHN=Pyv Resin

Fmoc-glycyl-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size

100-200 mesh

Loading > 0.3 mmol/g

DVB 1% DVB

PYV1180

Fmoc-L-His(Trt)-NHN=Pyv Resin

Fmoc-N-trityl-L-histidyl-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size 100-200 mesh

Loading > 0.3 mmol/g

DVB 1% DVB

PYV1190

Fmoc-L-Ile-NHN=Pyv Resin

Fmoc-L-isoleucyl-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size

100-200 mesh

Loading > 0.3 mmol/g

DVB 1% DVB

PYV1200

Fmoc-L-Leu-NHN=Pyv Resin

Fmoc-L-leucyl-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size 100-200 mesh

Loading > 0.3 mmol/g

DVB 1% DVB

PYV1210

Fmoc-L-Lys(Boc)-NHN=Pyv Resin

Fmoc-N-epsilon-t-butyloxycarbonyl-L-lysyl-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size

100-200 mesh

Loading > 0.3 mmol/g

DVB 1% DVB

PYV1220

Fmoc-L-Met-NHN=Pyv Resin

Fmoc-L-methionyl-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size 100-200 mesh

Loading > 0.3 mmol/g

DVB 1% DVB

PYV1230 Fmoc-L-Phe-NHN=Pyv Resin

Fmoc-L-phenylalanyl-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size 100-200 mesh

Loading > 0.3 mmol/g

DVB 1% DVB

PYV1240

Fmoc-L-Pro-NHN=Pyv Resin

Fmoc-L-prolinyl-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size 100-200 mesh

Loading > 0.3 mmol/g

DVB 1% DVB

PYV1250

Fmoc-L-Ser(t Bu)-NHN=Pyv Resin

Fmoc-O-t-butyl-L-seryl-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size

100-200 mesh

Loading > 0.3 mmol/g

DVB 1% DVB

PYV1260

Fmoc-L-Thr(t Bu)-NHN=Pyv Resin

Fmoc-O-t-butyl-L-threonyl-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size

100-200 mesh

Loading > 0.3 mmol/g

DVB 1% DVB

PYV1270

Fmoc-L-Trp(Boc)-NHN=Pyv Resin

Fmoc-N-t-butyloxycarbonyl-L-tryptophyl-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size

100-200 mesh

Loading > 0.3 mmol/g

DVB 1% DVB

Cyclic Peptides

PYV1280 Fmoc-L-Tyr(t Bu)-NHN=Pyv Resin

Fmoc-O-t-butyl-L-tyrosyl-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size

100-200 mesh

Loading > 0.3 mmol/g

DVB 1% DVB

PYV1290 Fmoc-L-Val-NHN=Pyv Resin

Fmoc-L-valyl-hydrazono-pyruvyl-aminomethylpolystyrene resin

Mesh Size

100-200 mesh

Loading > 0.3 mmol/g DVB 1% DVB

References:

→ Facile and efficient chemical synthesis of APET×2, an ASIC-targeting toxin, via hydrazide-based native chemical ligation; S.-J. Li, D.-L. Qu, Y.-H. Wang, Y. He, M. Wen, Q.-X. Guo, J. Shi, Y.-M. Li; Tetrahedron 2015; 71: 3363-3366. arrow-up-right-from-square https://doi.org/10.1016/j.tet.2015.03.098

→ Chemical synthesis of proteins using peptide hydrazides as thioester surrogates; J.-S. Zheng, S. Tang, Y.-K. Qi, Z.-P. Wang, L. Liu; Nature Protocols 2013; 8: 2483. arrow-up-right-from-square https://doi.org/10.1038/nprot.2013.152

→ 44. Amino-oxy-derivatives. Part I. Some a-amino-oxy-acids and a-amino-oxy-hydrazides. D. Mchale, J. Green, P. Mamalis; J. Chem. Soc. 1960; 225-229. arrow-up-right-from-square https://doi.org/10.1039/JR9600000225

→ SAR by Oxime-Containing Peptide Libraries: Application to Tsg101 Ligand Optimization; F. Liu, A. G. Stephen, A. A. Waheed, M. J. Aman, E. O. Freed, R. J. Fisher, T. R. Burke; ChemBioChem 2008; 9(12) : 2000-2004. arrow-up-right-from-square https://doi.org/10.1002/cbic.200800281

→ A Versatile Set of Aminooxy Amino Acids for the Synthesis of Neoglycopeptides; M. R. Carrasco, R. T. Brown; J. Org. Chem. 2003; 68: 8853-8858. arrow-up-right-from-square https://doi.org/10.1021/jo034984x

→ Oxime Ligation: A Chemoselective Click-Type Reaction for Accessing Multifunctional Biomolecular Constructs; S. Ulrich, D. Boturyn, A. Marra, O. Renaudet, P. Dumy; Chem. Eur. J. 2013; 20(1) : 34-41. arrow-up-right-from-square https://doi.org/10.1002/chem.201302426

4.1.2. Azide-Alkyne Cycloaddition-Mediated Peptide Cyclization/Click Cyclization

Alkynes and azides can undergo a Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) to afford 1,4-disubstituted 1,2,3-triazoles. Developed by K. Barry Sharpless and Morton Meldal, this type of chemical transformation was quickly dubbed “Click chemistry”. It has since become a widely used reaction that is orthogonal to many other types of chemical transformations and is used in various kinds of applications. Due to its high thermodynamic driving force, which is usually greater than 20 kcal/mol, the Click reaction rapidly proceeds to completion in almost all cases. CuAAC is highly selective for the 1,4-disubstituted 1,2,3-triazole, whereas ruthenium catalysis affords the 1,5-disubstituted product.

+ N3 Cu(I) Ru(II) N N N N N N

Fig. 12: Schematic illustration of the azide-alkyne-cycloaddition, either Cu(I)- or Ru(II)-catalyzed.

Iris Biotech offers a variety of azido and alkyne amino acids. They can be incorporated into biomolecules by recombinant syntheses, particularly by non-neutral protein translation using the amber-suppressionbased orthogonal system, or by chemical reactions. The reaction conditions are fully compatible with SPPS and the reaction could be performed on side-chain unprotected peptides, displaying the high chemoselectivity of the reaction between alkynes and azides.

The resultant 1,4-disubstituted 1,2,3-triazole was found to effectively mimic the native trans-amide bond, which provided peptide chemists with another option of amide bond isosteres. In contrast, the 1,5-disubstituted product provided by ruthenium(II) catalysis is a good peptidomimetic of the cis-amide bond.

For ease of synthesis, the cyclization of peptides by Click chemistry mainly involves modified side-chains instead of the termini as the introduction of side-chain modified amino acids is very convenient and many building blocks are readily commercially available.

4.1.3. Aziridine Aldehyde-Based Multicomponent Macrocyclization

Yudin and co-workers have developed an Ugi-typed four-component-based macrocyclization. This method involved an aziridine aldehyde, an isocyanide, the N-terminal amino group, and the C-terminal carboxylic acid of a peptide segment.

Fig. 13: Schematic illustration of the aziridine aldehyde-based multicomponent macrocyclization.

Cyclic Peptides

Reference:

→ Mechanistic investigation of aziridine aldehyde-driven peptide macrocyclization: the imidoanhydride pathway; S. Zaretsky, J. L. Hickey, J. Tan, D. Pichugin, M. A. St. Denis, S. Ler, B. K. W. Chung, C. C. G. Scully, A. K. Yudin; Chem. Sci. 2015; 6: 5446-5455. arrow-up-right-from-square https://doi.org/10.1039/C5SC01958C

4.1.4. Imine-Mediated Macrocyclization

Baran and co-workers developed a peptide macrocyclization method utilizing the N-terminal amino group and the C-terminal aldehyde of an unprotected linear peptide. Like many other cyclization strategies, this cyclization had to be performed at highly diluted conditions (1 mM). This peptide cyclization started with spontaneous intramolecular imine formation in aqueous media, followed by the nucleophilic attack of the imine to “trap” the cyclic peptide. Depending on the choice of nucleophiles, different moieties at the cyclization site were formed. External nucleophiles such as KCN and NaBH 3 CN generated α-aminonitriles or secondary amines at the ring-closure site, respectively, while internal nucleophiles adjacent to the N-terminal amino group, including indole, imidazole, thiol, and selenol, generated heterocycles at the cyclization site.

Fig. 14: Schematic illustration of the imine-mediated macrocyclization

The beauty of this cyclization technology is the ability to introduce diverse modifications after cyclization, which enables the generation of an array of analogues derived from the same linear precursors facilitating the synthesis of whole peptide libraries.

Reference:

→ Peptide Macrocyclization Inspired by Non-Ribosomal Imine Natural Products; L. R. Malins, J. N. deGruyter, K. J. Robbins, P. M. Scola, M. D. Eastgate, M. Reza Ghadiri, P. S. Baran; J. Am. Chem. Soc. 2017; 139(14): 5233-5241. arrow-up-right-from-square https://doi.org/10.1021/jacs.7b01624

4.1.5. Cyanopyridines and Aminothiols for Peptide Cyclization

Cyanopyridine click-like reactions may be used for macrocyclization, stapling, bi- and even tricyclization. The required 1,2-aminothiols can be provided in three ways: a) as N-terminal cysteine (NCys), b) as intra-chain 1,2 aminothiol or c) as 1,3-thiazolidine which can be deprotected later in situ In vitro, the reaction is optimally carried out at a pH between 7 and 8. In recombinant proteins, N-terminal cysteines may be provided by the action of TEV protease.

With a 2,6 dicyanopyridine (2,6-DCP) side-chain as bidentate anchor, the formation of bicyclic peptides is rather simple, just two 1,2-aminothiols need to be provided. The cyclization itself will take place within a few minutes in the presence of TCEP (tris-carboxyethylphosphine; LS-3405: arrow-up-right-from-square https://www. iris-biotech.de/ls-3405) as reductant, in a neutral buffer at pH 7.5 and ambient temperature.

Fig. 15: Example for the synthesis of a bicyclic peptide: When a peptide with an N-terminal cysteine, a C-terminal 1,2-aminothiol and an intra-chain dicyanopyridine (2,6-DCP) is incubated for a few minutes at aqueous neutral conditions, the reaction between the 2,6-DCP and the 1,2 aminothiols will take place quickly. TCEP serves as reduction agent and ensures that the thiols remain in their reduced form.

2,6-Dicyanopyridine may also be used in a non-bound form to make cyclic peptides, when the two 1,2aminothiols are provided as side-chains, introduced as non-canonical amino acids. For the controlled synthesis of tricyclic peptides, this method even may be combined with the synthesis of bicyclic peptides, when two more 1,2-aminothiols initially are introduced in a protected fashion as 1,3 thiazoles, are deprotected with methoxyamine after the first cyclization and the 2,6-DCP is used to form the thiazoline bridge.

References:

→ The Cyanopyridine-Aminothiol Click Reaction: Expanding Horizons in Chemical Biology; C. Nitsche; SynLett. 2024; 35: A-E. arrow-up-right-from-square https://dx.doi.org/10.1055/a-2214-7612

→ Biocompatible and Selective Generation of Bicyclic Peptides; S. Ullrich, J. George, A. Coram, R. Morewood, C. Nitsche; Angew. Chem Int. Ed. 2022; 61(43): e20228400. arrow-up-right-from-square https://doi.org/10.1002/anie.202208400

→ Tobacco Etch Virus protease: A shortcut across biotechnologies; F. Cesaratto, O. Burrone, G. Petris; J Biotechnol. 2016; 231(10) : 239-249. arrow-up-right-from-square https://doi.org/10.1016/j.jbiotec.2016.06.012

Cyclic Peptides

FAA9370 Fmoc-L-3-(2-cyano-4-pyridyl)-alanine

(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2cyanopyridin-4-yl)propanoic acid

CAS-No. 2245755-79-5

Formula C 24H19 N3 O4

Mol. weight 413,43 g/mol

FAA9375 Fmoc-L-3-(2-cyano-3-pyridyl)-alanine

(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2cyanopyridin-3-yl)propanoic acid

CAS-No. 2245755-77-3

Formula C 24H19 N3 O4

Mol. weight 413,43 g/mol

FAA9380 Fmoc-L-Dap(2-CINA)-OH

(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl) amino)-3-(2-cyanoisonicotinamido)propanoic acid (S)

Formula C 25H20 N4O 5 Mol. weight 456,46 g/mol

FAA9385 Fmoc-L-Dap(6-CNA)-OH

(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl) amino)-3-(6-cyanonicotinamido)propanoic acid

Formula C 25H20 N4O 5 Mol. weight 456,46 g/mol

FAA9390 Fmoc-L-Dab(2,6-DCP)-OH

(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4((2,6-dicyanopyridin-4-yl)amino)butanoic acid

CAS-No. 2968514-51-2

Formula C 26 H21N 5 O4 Mol. weight 467,49 g/mol

FAA9395 Fmoc-L-Cys(2,6-DCP)-OH

N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(2,6-dicyanopyridin-4-yl)-L-cysteine (R)

CAS-No. 2968514-50-1

Formula C 25H18 N4O4 S Mol. weight 470,50 g/mol

(S)

FAA9315 Fmoc-L-Lys(Boc-Cys(Trt))-OH

N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(N-(tert-butoxycarbonyl)-S-trityl-L-cysteinyl)-L-lysine

CAS-No. 587854-43-1

Formula C 48 H 51N3 O 7S Mol. weight 814,01 g/mol

FAA9320 Fmoc-L-Lys(Boc-Thz)-OH

N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-((R)-3-(tert-butoxycarbonyl)thiazolidine-4-carbonyl)-L-lysine

Formula C 30 H37N3 O 7S Mol. weight 583,70 g/mol

FAA9325 Fmoc-L-Dab(Boc-Cys(Trt))-OH

(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl) amino)-4-((R)-2-((tert-butoxycarbonyl)amino)-3-(tritylthio)propanamido)butanoic acid

CAS-No. 2968514-52-3

Formula C 46 H47N3 O 7S Mol. weight 785,96 g/mol

FAA9330 Fmoc-L-Dab(Boc-Thz)-OH

(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl) amino)-4-((R)-3-(tert-butoxycarbonyl)thiazolidine-4-carboxamido)butanoic acid

CAS-No. 2968514-54-5

Formula C 28 H33 N3 O 7S Mol. weight 555,65 g/mol

FAA9335 Fmoc-L-Lys(5-STrt, Boc)-OH

(2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl) amino)-6-((tert-butoxycarbonyl)amino)-5-(tritylthio) hexanoic acid

CAS-No. 1240666-29-8

Formula C 45H46 N2O 6 S Mol. weight 742,93 g/mol

FAA9340 Fmoc-L-Lys(4-Thz, Boc)-OH

(2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl) amino)-4-(3-(tert-butoxycarbonyl)thiazolidin-5-yl) butanoic acid

CAS-No. 1240666-28-7

Formula C 27H32N2O 6 S Mol. weight 512,62 g/mol

Cyclic Peptides

4.2. Peptide Linkages

4.2.1. Thiazolidine-Generated Cyclization

With this method, cyclic peptides with ring sizes from five to 26 residues were successfully synthesized. Although no limitations for the C-terminal amino acid are reported, all synthesized model peptides reported by Tam et al. contained Gly-cysteine at the cyclization sites. The intramolecular cyclization is initiated in a pH 5.5 aqueous solution followed by O -to-N-acyl transfer at pH 5.9. Heating promotes the O -to-N-acyl transfer, but as side reactions such as hydrolysis and decomposition are observed. The rate of the cyclization is dependent on the formed ring size – the larger, the faster.

Fig. 16: Schematic illustration of the thiazolidine-generated cyclization.

Reference:

→ Cyclic Peptides from Linear Unprotected Peptide Precursors through Thiazolidine Formation; P. Botti, T. D. Palinn, J. P. Tam; J. Am. Chem. Soc. 1996; 118(42) : 1018-10024. arrow-up-right-from-square https://doi.org/10.1021/ja954278g

4.2.2. Native Chemical Ligation-Mediated Cyclization

Native chemical ligation (NCL) of unprotected peptide segments involves the reaction between a first peptide fragment α-thioester and a second peptide fragment which carries a cysteine on the N-terminus, to yield a product with a native amide bond at the ligation site. NCL can also be utilized for synthesizing cyclic peptides. As alternative, the N-terminal cysteine can be replaced by selenocysteine.

Fig. 16: Schematic illustration of native chemical ligation.

FAA8600 Fmoc-D-Sec(Xan)-OH

Fmoc-Se-xanthyl-D-selenocysteine

CAS-No. 2988660-46-2

Formula C 31H25NO 5 Se Mol. weight 570,49 g/mol

HAA9255 H-L-Sec(o Nv)-OH*TFA

Dimethoxynitrobenzyl selenocysteine TFA salt

CAS-No. 1644398-13-9

Formula C12H16 N2O 6 Se*CF 3 COOH Mol. weight 363,24*114,02 g/mol

HAA9360 H-L-Sec(MDNPE)-OH

Se-(Methyl-o-nitropiperonyl)-selenocysteine

CAS-No. 2235373-47-2

Formula C12H14N2O 6 Se Mol. weight 361,21 g/mol

FAA8705 Fmoc-L-Sec(Mob)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-Se-(4-methoxybenzyl)-L-selenocysteine

CAS-No. 150308-80-8

Formula C 26 H25NO 5 Se Mol. weight 510,46 g/mol

FAA8465 Fmoc-L-Sec(Xan)-OH

Fmoc-Se-xanthyl-L-selenocysteine

CAS-No. 1639843-35-8

Formula C 31H25NO 5 Se Mol. weight 570,49 g/mol

FAA8760 Fmoc-L-Sec(Mob)-OPfp

N-alpha-(9-Fluorenylmethyloxycarbonyl)-L-4-methoxybenzyl selenocysteine pentafluorophenyl ester

CAS-No. 939431-43-3

Formula C 32H24F5NO 5 Se Mol. weight 676,51 g/mol

Cyclic Peptides

BAA4830 Boc-L-Sec(Mob)-OPfp

N-alpha-tert-Butoxycarbonyl-4-methoxybenzyl-L-selenocysteine pentafluorophenyl ester

CAS-No. 1257525-48-6

Formula C 22H22F5NO 5 Se Mol. weight 554,38 g/mol

FAA8710 Fmoc-D-Sec(Mob)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-Se-(4-methoxybenzyl)-D-selenocysteine

CAS-No. 2987041-11-0

Formula C 26 H25NO 5 Se Mol. weight 510,46 g/mol

HAA9230 H-L-Sec(MDNPE)*TFA

(2R)-2-amino-3-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl) ethyl)selanyl)propanoic acid trifluoroacetate

CAS-No. 2235373-48-3

Formula C12H14N2O 6 Se*CF 3 CO 2H Mol. weight 361,22*114,02 g/mol

BAA3760 Boc-L-Sec(Mob)-OH

N-alpha-t-Butyloxycarbonyl-Se-(4-methoxybenzyl)-L-selenocysteine

CAS-No. 959415-39-5

Formula C16 H23 NO 5 Se Mol. weight 388,32 g/mol

HAA9465 H-L-Sec(oNB)-OH*HCl

(R)-2-amino-3-((2-nitrobenzyl)selanyl)propanoic acid

CAS-No. 324582-23-2 net

Formula C10 H12N2O4 Se*HCl Mol. weight 303,18*36,46 g/mol

HAA9475 H-L-Sec(NPE)-OH*HCl

(2R)-2-amino-3-((1-(2-nitrophenyl)ethyl)selanyl)propanoic acid

Formula C11H14N2O4 Se*HCl Mol. weight 317,02*36,46 g/mol

Besides Sec, our portfolio contains a variety of selenazolidine carboxylic acids (Sez derivatives). Sez can be deprotected and converted to Sec by treatment with O -methylhydroxylamine (MeONH2) at pH 4 or by using Cu(II) salts. Product details

BAA4880 Boc-L-Sez-OH

Boc selenazolidine carboxylic acid

CAS-No. 1841180-44-6

Formula C9 H15NO4 Se Mol. weight 280,19 g/mol

FAA8860 Fmoc-L-Sez-OH

Fmoc selenazolidine carboxylic acid

CAS-No. 1985651-74-8

Formula C19 H17NO4 Se

Mol. weight 402,31 g/mol

4.2.3. Traceless Staudinger Ligation-Mediated Peptide Cyclization

Staudinger ligation describes the chemoselective reaction between azides and triaryl phosphines or N-acylimidazole phosphines to generate an amide. Kleineweischede and Hackenberger first described that this type of reaction can be utilized for the synthesis of cyclic peptides starting from side-chain unprotected linear peptides.

Fig. 17: Schematic illustration of the traceless Staudinger ligation-mediated peptide cyclization.

Reference:

→ Chemoselective Peptide Cyclization by Traceless Staudinger Ligation; R. Kleineweischede, C. P. R. Hackenberger; Angew. Chem. Int. Ed. 2008; 47(32): 5984-5988. arrow-up-right-from-square https://doi.org/10.1002/anie.200801514

Cyclic Peptides

4.2.4. Alpha-Ketoacid Hydroxylamine Ligation-Mediated Peptide Cyclization

The chemoselective reaction between a C-terminal α-ketoacid (KA) and an N-terminal hydroxyl-amine (HA) for the formation of an amide bond at the ligation site is reported for the preparation of headto-tail cyclic peptides. Eventhough this technology can be used for cyclization at any site within the peptide, it is rarely used, most likely due to the low stability of the free hydroxylamines and the need of temporary protection of the N-terminus during precursor synthesis. Advances like the development of the stable 5-oxaproline as N-terminal hydroxylamine and the protected α-ketoacid resins make the approach more convenient, however, noticeable epimerization at the C-terminal residue remains a major drawback.

Fig. 18: Schematic illustration of the KAHA ligation.

References:

→ Chemoselective Amide Ligations by Decarboxylative Condensations of N-Alkylhydroxylamines and alphaKetoacids; J. W. Bode, R. M. Fox, K. D. Baucom; Angew. Chem. Int. Ed. 2006; 45(8): 1248-1252. arrow-up-right-from-square https://doi.org/10.1002/anie.200503991

→ Stereoretentive synthesis and chemoselective amide-forming ligations of C-terminal peptide alpha-ketoacids; L. Ju, A. R. Lippert, J. W. Bode; J. Am. Chem. Soc. 2008; 130(13): 4253-5. arrow-up-right-from-square https://doi.org/10.1021/ja800053t

→ Chemical Protein Synthesis with the alpha-Ketoacid-Hydroxylamine Ligation; J. W. Bode; Acc. Chem. Res. 2017; 50(9): 2104-2115. arrow-up-right-from-square https://doi.org/10.1021/acs.accounts.7b00277

→ Chemical protein synthesis by chemoselective-alpha-ketoacid-hydroxylamine (KAHA) ligations with 5-oxaproline; V. R. Pattabiraman, A. O. Ogunkoya, J. W. Bode; Angew. Chem. Int. Ed. Engl. 2012; 51(21) : arrow-up-right-from-square https://doi.org/10.1002/anie.201200907

4.2.5. Serine/Threonine Ligation-Mediated Peptide Cyclization

Oxazolidine formation via reaction of the 1,2-hydroxy amine bifunctional groups of an N-terminal serine or threonine residue of an unprotected peptide with a C-terminal peptide salicylaldehyde ester and subsequent O -to-N-acyl transfer followed by acidolysis provides the natural peptide Xaa-Ser/Thr linkage at the ligation site.

Fig. 19: Schematic illustration of serine/threonine ligation-mediated peptide cyclization.

References:

→ Serine/Threonine Ligation: Origin, Mechanistic Aspects, and Applications; H. Li, X. Li; Acc. Chem. Res. 2018; 51(7) : 1643-1655. arrow-up-right-from-square https://doi.org/10.1021/acs.accounts.8b00151

→ Protein chemical synthesis by serine and threonine ligation; Y. Zhang, C. Xu, H. Y. Lam, C. L. Lee, X. Li; PNAS 2013; 110(17): 6657-6662. arrow-up-right-from-square https://doi.org/10.1073/pnas.1221012110

→ Salicylaldehyde ester-induced chemoselective peptide ligations: enabling generation of natural peptidic linkages at the serine/threonine sites; X. Li, H. Y. Lam, Y. Zhang, C. K. Chan; Org. Lett. 2010; 12(8): 1724-7. arrow-up-right-from-square https://doi.org/10.1021/ol1003109

4.3. Enzyme-Mediated Peptide Cyclization

Enzyme-mediated ligation is providing an alternative to chemical synthesis for the cyclization step. Enzyme activities are highly chemoselective. Their nontoxic and catalytic properties are of great value to the pharmaceutical industry in the preparation of cyclic peptide drugs considering purity and cost-effectiveness. Examples include Sortase A, Butelase 1, GmPOPB, and peptiligase.

Reference:

→ Ligation Technologies for the Synthesis of Cyclic Peptides; H. Y. Chow, Y. Zhang, E. Matheson, X. Li; Chem. Rev. 2019; 119(17) : 9971-10001; arrow-up-right-from-square https://doi.org/10.1021/acs.chemrev.8b00657 . circle-arrow-right

For more information on Ligation Technologies, download our brochure!

Cyclic Peptides

5. Product Overview

5.1. Cysteine Building Blocks

AAA1300 Ac-L-Cys-OH

N-alpha-Acetyl-L-cysteine

CAS-No. 616-91-1

Formula C 5H9 NO 3 S Mol. weight 163,19 g/mol

FAA3340 Fmoc-L-MeCys(S-t Bu)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-N-alpha-methyl-S-(t-butylthio)-L-cysteine

CAS-No. 1013096-03-1

Formula C 23 H27NO4 S 2 Mol. weight 445,59 g/mol

HAA1017 H-D-Cys-OH*HCl*H2O

D-Cysteine hydrochloride monohydrate

CAS-No. 32443-99-5

Formula C 3 H7NO 2 S*HCl*H2O Mol. weight 121,2*36,45*18,01 g/mol

HAA6070 H-L-Cys(Acm)-OH*HCl

S-(Acetyl-aminomethyl)-L-cysteine hydrochloride

CAS-No. 28798-28-9

Formula C 6 H12N2O 3 S*HCl Mol. weight 192,24*36,45 g/mol

HAA1574 H-L-Cys(Bzl)-OH

S-Benzyl-L-cysteine

CAS-No. 3054-01-1

Formula C10 H13 NO 2 S Mol. weight 211,29 g/mol

HAA6090 H-L-Cys(MBzl)-OH

S-(4-Methylbenzyl)-L-cysteine

CAS-No. 42294-52-0

Formula C11H15NO 2 S Mol. weight 225,3 g/mol

HAA6110 H-D-Cys(Bzl)-OH

S-Benzyl-D-cysteine

CAS-No. 23032-53-3

Formula C10 H13 NO 2 S Mol. weight 211,29 g/mol

HAA6080 H-L-Cys(Bzl)-OMe*HCl

S-Benzyl-L-cysteine methyl ester hydrochloride

CAS-No. 16741-80-3

Formula C11H15NO 2 S*HCl Mol. weight 225,31*36,45 g/mol

HAA1078 H-L-Cys(Me)-OH*HCl

S-Methyl-L-cysteine hydrochloride

CAS-No. 13331-74-3

Formula C 4H9 NO 2 S*HCl Mol. weight 135,19*36,45 g/mol

HAA2350 H-L-Cys(Propargyl)-OH*HCl

S-Propargyl-L-cysteine hydrochloride

CAS-No. 3262-64-4

Formula C 6 H9 NO 2 S*HCl Mol. weight 159,21*36,45 g/mol

HAA9270 H-L-Cys(MDNPE)-OH

1-[4‘,5‘-(methylenedioxy)-2‘-nitrophenyl]ethyl]-L-cysteine

CAS-No. 1551078-43-3

Formula C12H14N2O 6 S Mol. weight 314,31 g/mol

Cyclic Peptides

HAA9320 H-L-Cys(o Nv)-OH

S-(4,5-dimethoxy-2-nitrobenzyl)-L-cysteine

CAS-No. 214633-68-8

Formula C12H16 N2O 6 S Mol. weight 316,33 g/mol

HAA6100 H-L-Cys(Mob)-OH

S-(4-Methoxybenzyl)-L-cysteine

CAS-No. 2544-31-2

Formula C11H15NO 3 S Mol. weight 241,3 g/mol

HAA6140 H-L-Cys(St Bu)-OH

S-Thio-t-butyl-L-cysteine

CAS-No. 30044-51-0

Formula C 7H15NO 2 S 2 Mol. weight 209,32 g/mol

HAA6150 H-L-Cys(t Bu)-OH*HCl

S-t-Butyl-L-cysteine hydrochloride

CAS-No. 2481-09-6

Formula C 7H15NO 2 S*HCl Mol. weight 177,26*36,45 g/mol

HAA1560 H-L-Cys(Trt)-NH2

S-Trityl-L-cysteine amide

CAS-No. 166737-85-5

Formula C 22H22N2OS Mol. weight 362,49 g/mol

HAA6160 H-L-Cys(Trt)-OH

S-Trityl-L-cysteine

CAS-No. 2799-07-7

Formula C 22H21NO 2 S Mol. weight 363,48 g/mol

HAA1995 H-L-Cys(Trt)-Ot Bu*HCl

S-Trityl-L-cysteine t-butyl ester hydrochloride

CAS-No. 158009-03-1

Formula C 26 H29 NO 2 S*HCl Mol. weight 419,58*36,45 g/mol

HAA2100 H-D-Cys(Trt)-Ot Bu*HCl

S-Trityl-D-cysteine t-butyl ester hydrochloride

CAS-No. 439089-10-8

Formula C 26 H29 NO 2 S*HCl Mol. weight 419,58*36,45 g/mol

HAA6120 H-D-Cys(Trt)-OH

S-Trityl-D-cysteine

CAS-No. 25840-82-8

Formula C 22H21NO 2 S Mol. weight 363,48 g/mol

HAA2810 N3 -L-Cys(Trt)-OH*CHA

(R)-2-azido-3-(tritylthio)propanoic acid cyclohexylamine

CAS-No. 1286670-90-3

Formula C 22H19 N3 O 2 S*C 6 H13 N Mol. weight 389,47*99,17 g/mol

BAA1083 Boc-L-Cys-OH

N-alpha-t-Butyloxycarbonyl-L-cysteine

CAS-No. 20887-95-0

Formula C 8 H15NO4 S Mol. weight 221,27 g/mol

BAA1170 Boc-D-Cys-OH

N-alpha-t-Butyloxycarbonyl-D-cysteine

CAS-No. 149270-12-2

Formula C 8 H15NO4 S Mol. weight 221,27 g/mol

Cyclic Peptides

BAA1078

Boc-L-Cys(Acm)-OH

N-alpha-t-Butyloxycarbonyl-S-(acetyl-aminomethyl)-L-cysteine

CAS-No. 19746-37-3

Formula C11H20 N2O 5 S Mol. weight 292,36 g/mol

BAA1510

Boc-L-Cys(Acm,O)-OH

N-alpha-t-Butyloxycarbonyl-S-(acetyl-aminomethyl)-S-oxo-L-cysteine

CAS-No. 75893-04-8

Formula C11H20 N2O 6 S Mol. weight 308,35 g/mol

BAA4360

Boc-L-Cys(CF3)-OH

N-alpha-t-Butyloxycarbonyl-S-trifluoromethyl-L-cysteine

CAS-No. 943926-18-9

Formula C9 H14F 3 NO4 S Mol. weight 289,27 g/mol

BAA1079

Boc-L-Cys(Bzl)-OH

N-alpha-t-Butyloxycarbonyl-S-benzyl-L-cysteine

CAS-No. 5068-28-0

Formula C15H21NO4 S Mol. weight 311,38 g/mol

BAA5410

Boc-D-Cys(Bzl)-OH

N-alpha-t-Butyloxycarbonyl-S-benzyl-D-cysteine

CAS-No. 102830-49-9

Formula C15H21NO4 S Mol. weight 311,38 g/mol

BAA5510

Boc-L-Cys(Fm)-OH

N-alpha-t-Butyloxycarbonyl-S-(9-fluorenylmethyl)-L-cysteineN-alpha-t-Butyloxycarbonyl-S-(9-fluorenylmethyl)-L-cysteine

CAS-No. 84888-35-7

Formula C 22H25NO4 S Mol. weight 399,51 g/mol

BAA1080 Boc-L-Cys(MBzl)-OH

N-alpha-t-Butyloxycarbonyl-S-(4-methyl-benzyl)-L-cysteine

CAS-No. 61925-77-7

Formula C16 H23 NO4 S Mol. weight 325,43 g/mol

BAA5420 Boc-D-Cys(MBzl)-OH

N-alpha-t-Butyloxycarbonyl-S-(4-methyl-benzyl)-D-cysteine

CAS-No. 61925-78-8

Formula C16 H23 NO4 S Mol. weight 325,43 g/mol

BAA1081 Boc-L-Cys(Mob)-OH

N-alpha-t-Butyloxycarbonyl-S-(4-methoxy-benzyl)-L-cysteine

CAS-No. 18942-46-6

Formula C16 H23 NO 5 S Mol. weight 341,43 g/mol

HAA3510 H-L-Cys(Npys)-OH*HCl

S-(3-nitro-2-pyridylthio)-L-cysteine hydrochloride

CAS-No. 108807-66-5

Formula C 8 H9 N3 O4 S 2*HCl Mol. weight 275,30*36,45 g/mol

BAA1860 Boc-L-Cys(Npys)-OH

N-alpha-t-Butyloxycarbonyl-S-(3-nitro-2-pyridylthio)-L-cysteine

CAS-No. 76880-29-0

Formula C13 H17N3 O 6 S 2 Mol. weight 375,42 g/mol

BAA2250 Boc-L-Cys(Propargyl)-OH*DCHA

N-alpha-t-Butyloxycarbonyl-S-propargyl-L-cysteine dicyclohexylamine

CAS-No. 1260119-25-2 net

Formula C11H17NO4 S*C12H23 N Mol. weight 259,32*181,32 g/mol

Cyclic Peptides

BAA3140 Boc-L-Cys(Ph)-OH

N-alpha-t-Butyloxycarbonyl-S-phenyl-L-cysteine

CAS-No. 163705-28-0

Formula C14H19 NO4 S Mol. weight 297,37 g/mol

BAA1082 Boc-L-Cys(t Bu)-OH

N-alpha-t-Butyloxycarbonyl-S-t-butyl-L-cysteine

CAS-No. 56976-06-8

Formula C12H23 NO4 S Mol. weight 277,37 g/mol

BAA1084 Boc-L-Cys(Trt)-OH

N-alpha-t-Butyloxycarbonyl-S-trityl-L-cysteine

CAS-No. 21947-98-8

Formula C 27H29 NO4 S Mol. weight 463,59 g/mol

BAA5000 Boc-D-Cys(Trt)-OH

N-alpha-t-Butyloxycarbonyl-S-trityl-D-cysteine

CAS-No. 87494-13-1

Formula C 27H29 NO4 S Mol. weight 463,59 g/mol

HAA3530 H-L-Cys-NH2*HCl

L-Cysteine amide hydrochloride

CAS-No. 16359-98-1

Formula C 3 H 8 N2OS*HCl Mol. weight 120,17*36,45 g/mol

FAA1362 Fmoc-L-Cys-OH*H2O

N-alpha-(9-Fluorenylmethyloxycarbonyl)-L-cysteine monohydrat

CAS-No. 135248-89-4

Formula C18 H17NO4 S*H2O Mol. weight 343,40*18,01 g/mol

FAA1470 Fmoc-D-Cys-OH*H2O

N-alpha-(9-Fluorenylmethyloxycarbonyl)-D-cysteine monohydrat

CAS-No. 157355-80-1

Formula C18 H17NO4 S*H2O Mol. weight 343,4*18,01 g/mol

FAA1980 Fmoc-L-Cys-NH2

N-alpha-(9-Fluorenylmethyloxycarbonyl)-L-cysteine amide

CAS-No. 623177-62-8

Formula C18 H18 N2O 3 S Mol. weight 342,41 g/mol

FAA1506 Fmoc-L-Cys(Acm)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(acetyl-aminomethyl)-L-cysteine

CAS-No. 86060-81-3

Formula C 21H22N2O 5 S Mol. weight 414,48 g/mol

FAA9250 Fmoc-L-Cys(2-Boc-aminoethyl)-OH

N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(2-((tert-butoxycarbonyl)amino)ethyl)-L-cysteine

CAS-No. 2230472-96-2

Formula C 25H30 N2O 6 S Mol. weight 486,58 g/mol

FAA9090 Fmoc-L-Cys(Cam)-OH

N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(2-amino-2-oxoethyl)-L-cysteine

CAS-No. 1443324-12-6

Formula C 20 H20 N2O 5 S Mol. weight 400,45 g/mol

FAA6910 Fmoc-L-Cys(Phacm)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-Phenylacetylaminomethyl)-L-cysteine

CAS-No. 159680-21-4

Formula C 27H26 N2O 5 S Mol. weight 490,57 g/mol

Cyclic Peptides

FAA6720 Fmoc-L-MeCys(Acm)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-N-alpha-methyl-S-(acetyl-aminomethyl)-L-cysteine

CAS-No. 481642-19-7

Formula C 22H24N2O 5 S Mol. weight 428,5 /mol

BAA6390 Boc-L-Cys(Phacm)-OH

N-alpha-t-Butyloxycarbonyl-S-(Phenylacetylaminomethyl)-L-cysteine

CAS-No. 57084-73-8

Formula C17H24N2O 5 S Mol. weight 368,45 g/mol

FAA3710 Fmoc-D-Cys(Phacm)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(phenylacetylaminomethyl)-D-cysteine

CAS-No. 1565818-55-4

Formula C 27H26 N2O 5 S Mol. weight 490,57 g/mol

FAA5150 Fmoc-L-Cys(Aapam)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-((4-(allyloxycarbonylamino)phenylacetylaminomethyl)-L-cysteine

CAS-No. 1946783-89-6

Formula C 31H31N3 O 7S Mol. weight 589,66 g/mol

FAA7610 Fmoc-L-Cys(Allocam)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-((allyloxycarbonylamino)methy)-L-cysteine

CAS-No. 232953-09-2

Formula C 23 H24N2O 6 S Mol. weight 456,51 g/mol

FAA6230 Fmoc-D-Cys(Acm)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(acetyl-aminomethyl)-D-cysteine

CAS-No. 168300-88-7

Formula C 21H22N2O 5 S Mol. weight 414,48 g/mol

FAA3720 Fmoc-L-Cys(Biotin)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-biotinyl-L-cysteine

Formula C 28 H31N3 O 6 S 2 Mol. weight 569,69 g/mol

FAA6270 Fmoc-L-Cys(Bzl)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-benzyl-L-cysteine

CAS-No. 53298-33-2

Formula C 25H23 NO4 S Mol. weight 433,52 g/mol

FAA1714 Fmoc-L-Cys(MBzl)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(4-methylbenzyl)-L-cysteine

CAS-No. 136050-67-4

Formula C 26 H25NO4 S Mol. weight 447,53 g/mol

FAA8225 Fmoc-L-Cys(CF3)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-trifluoromethyl-L-cysteine

CAS-No. 1994331-25-7

Formula C19 H16 F 3 NO4 S Mol. weight 411,4 g/mol

FAA1030 Fmoc-L-Cys(Mmt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-p-methoxytrityl-L-cysteine

CAS-No. 177582-21-7

Formula C 38 H33 NO 5 S Mol. weight 615,74 g/mol

FAA4845 Fmoc-alpha-Me-L-Cys(Mmt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-alpha-methyl-S-(4-methoxytrityl)-L-cysteine

CAS-No. 1198791-74-0

Formula C 39 H35NO 5 S Mol. weight 629,76 g/mol

Cyclic Peptides

HAA3500 H-D-Cys(Mmt)-OH

S-p-methoxytrityl-D-cysteine

CAS-No. 926935-33-3

Formula C 23 H23 NO 3 S Mol. weight 393,5 g/mol

FAA1614 Fmoc-D-Cys(Mmt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-p-methoxytrityl-D-cysteine

CAS-No. 1198791-73-9

Formula C 38 H33 NO 5 S Mol. weight 615,74 g/mol

FAA1715 Fmoc-L-Cys(Mob)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(4-methoxybenzyl)-L-cysteine

CAS-No. 141892-41-3

Formula C 26 H25NO 5 S Mol. weight 463,55 g/mol

FAA8410 Fmoc-L-Cys(SO2Mob)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(4-methoxybenzyl)-L-cysteine-S,S-dioxide

CAS-No. 2412536-40-2

Formula C 26 H25NO 7S Mol. weight 495,55

FAA4155 Fmoc-L-Cys(Msbh)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(4,4‘-dimethylsulfinylbenzhydryl)-L-cysteine

CAS-No. 1584646-97-8

Formula C 33 H31NO 6 S 3 Mol. weight 633,80 g/mol

FAA8495 Fmoc-L-Cys(SIT)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(sec-isoamyl mercaptan)-L-cysteine

CAS-No. 2545642-31-5

Formula C 23 H27NO4 S 2 Mol. weight 445,59 g/mol

FAA4160 Fmoc-L-Cys(Thp)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-tetrahydropyranyl-L-cysteine

CAS-No. 1673576-83-4

Formula C 23 H25NO 5 S Mol. weight 427,15 g/mol

FAA3810 Fmoc-L-Cys(Propargyl)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-propargyl-L-cysteine

CAS-No. 1354752-76-3

Formula C 21H19 NO4 S Mol. weight 381,44 g/mol

FAA1575 Fmoc-L-Cys(St Bu)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(t-butylthio)-L-cysteine

CAS-No. 73724-43-3

Formula C 22H25NO4 S 2 Mol. weight 431,57 g/mol

FAA3180 Fmoc-L-Cys(S-DMP)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(2,6-dimethoxythiophenol)-L-cysteine

CAS-No. 1403834-73-0

Formula C 26 H25NO 6 S 2 Mol. weight 511,61 g/mol

FAA1965 Fmoc-D-Cys(St Bu)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(t-butylthio)-D-cysteine

CAS-No. 501326-55-2

Formula C 22H25NO4 S 2 Mol. weight 431,57 g/mol

FAA1716 Fmoc-L-Cys(t Bu)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-t-butyl-L-cysteine

CAS-No. 67436-13-9

Formula C 22H25NO4 S Mol. weight 399,51 g/mol

Cyclic Peptides

HAA3520 H-D-Cys(Trt)-OMe*HCl

S-trityl-D-cysteine methyl ester hydrochloride

CAS-No. 1020369-32-7

Formula C 23 H23 NO 2 S*HCl Mol. weight 377,50*36,45 g/mol

FAA1040 Fmoc-L-Cys(Trt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-trityl-L-cysteine

CAS-No. 103213-32-7

Formula C 37H31NO4 S Mol. weight 585,71 g/mol

FAA5670 Fmoc-L-Cys(Trt)-OMe

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-trityl-L-cysteine methyl ester

CAS-No. 245088-56-6

Formula C 38 H33 NO4 S Mol. weight 599,74 g/mol

FAA3190 Fmoc-L-Cys(Dpm)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-diphenylmethyl-L-cysteine

CAS-No. 247595-29-5

Formula C 31H27NO4 S Mol. weight 509,62 g/mol

FAA5650 Fmoc-D-Cys(Dpm)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-diphenylmethyl-D-cysteine

CAS-No. 2389078-16-2

Formula C 31H27NO4 S Mol. weight 509,62 g/mol

FAA1035 Fmoc-D-Cys(Trt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-trityl-D-cysteine

CAS-No. 167015-11-4

Formula C 37H31NO4 S Mol. weight 585,71 g/mol

FAA6940 Fmoc-L-Cys(Ddm)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-di(4-methoxyphenyl)methyl-L-cysteine

CAS-No. 1403825-56-8

Formula C 33 H31NO 6 S Mol. weight 569,67 g/mol

FAA3570 Fmoc-L-MeCys(Trt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-N-alpha-methyl-S-trityl-L-cysteine

CAS-No. 944797-51-7

Formula C 38 H33 NO4 S Mol. weight 599,74 g/mol

FAA1950 Fmoc-L-Cys(Palm)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-t-palmitoyl-L-cysteine

CAS-No. 824955-27-3

Formula C 34H47NO 5 S Mol. weight 581,81 g/mol

FAA5890 Fmoc-L-Cys(Octyl)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-octyl-L-cysteine

CAS-No. 210883-65-1

Formula C 26 H33 NO4 S Mol. weight 455,61 g/mol

FAA4810 Fmoc-L-Cys(lauryl)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-dodecyl-L-cysteine

CAS-No. 1310682-09-7

Formula C 30 H41NO4 S Mol. weight 511,72 g/mol

FAA8770 Fmoc-L-Cys(palmityl)-OH

N-alpha-(9-Fluorenylmethoxycarbonyl)-S-hexadecyl-L-cysteine

CAS-No. 876312-48-0

Formula C 34H49 NO4 S Mol. weight 567,83 g/mol

Cyclic Peptides

FAA4820 Fmoc-L-Cys(Ph)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-phenyl-L-cysteine

CAS-No. 488761-06-4

Formula C 24H21NO4 S Mol. weight 419,49 g/mol

FAA4751 Fmoc-L-Cys(Ac-Ot Bu)-OH*DCHA

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(t-butoxycarbonylmethyl)-L-cysteine dicyclohexylamine

CAS-No. 269730-62-3 net

Formula C 24H27NO 6 S*C12H23 N Mol. weight 457,54*181,32 g/mol

FAA4760 Fmoc-L-Cys(EtCO-Ot Bu)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(t-butoxycarbonylethyl)-L-cysteine

CAS-No. 685863-48-3

Formula C 25H29 NO 6 S Mol. weight 471,57 g/mol

FAA3370 Fmoc-L-Cys(PrCO-Ot Bu)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(t-butoxycarbonylpropyl)-L-cysteine

CAS-No. 102971-73-3

Formula C 26 H31NO 6 S Mol. weight 485,59 g/mol

ZAA1161 Z-L-Cys(Bzl)-OH

N-alpha-Benzyloxycarbonyl-S-benzyl-L-cysteine

CAS-No. 3257-18-9

Formula C18 H19 NO4 S Mol. weight 345,42 g/mol

ZAA1310 Z-L-Cys(Trt)-OH

N-alpha-Benzyloxycarbonyl-S-trityl-L-cysteine

CAS-No. 26311-04-6

Formula C 30 H27NO4 S Mol. weight 497,60 g/mol

FAA4840 Fmoc-alpha-Me-L-Cys(Trt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-alpha-methyl-S-trityl-L-cysteine

CAS-No. 725728-43-8

Formula C 38 H33 NO4 S Mol. weight 599,74 g/mol

FAA4850 Fmoc-alpha-Me-D-Cys(Trt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-alpha-methyl-S-trityl-D-cysteine

CAS-No. 725728-37-0

Formula C 38 H33 NO4 S Mol. weight 599,74 g/mol

FAA3970 Fmoc-L-Cys(o Nv)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(2-nitroveratryl)-L-cysteine

CAS-No. 214633-71-3

Formula C 27H26 N2O 8 S Mol. weight 538,57 g/mol

FAA8420 Fmoc-L-Cys(NDBF)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(1-(3-nitro-dibenzofuran-2-yl)-ethyl)-L-cysteine

CAS-No. 1895883-28-9

Formula C 32H26 N2O 7S Mol. weight 582,62 g/mol

SAA1110 Smoc-L-Cys(Trt)-OH

N-(((2,7-disulfo-9H-fluoren-9-yl)methoxy)carbonyl)-S-trityl-L-cysteine potassium salt

CAS-No. 2442552-68-1

Formula C 37H29 K 2NO 10 S 3 Mol. weight 822,01 g/mol

Cyclic Peptides

5.2. Homocysteine Building Blocks

FAA8255 Fmoc-L-HCys(Acm)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(acetyl-aminomethyl)-L-homocysteine

CAS-No. 150281-21-3

Formula C 22H24N2O 5 S Mol. weight 428,5 g/mol

FAA8260 Fmoc-D-HCys(Acm)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(acetyl-aminomethyl)-D-homocysteine

CAS-No. 2576507-96-3

Formula C 22H24N2O 5 S Mol. weight 428,5 g/mol

BAA5180 Boc-D-HCys(MBz)-OH

N-alpha-t-Butyloxycarbonyl-S-(4-methylbenzyl)-D-homocysteine

CAS-No. 214630-13-4

Formula C17H25NO4 S Mol. weight 339,46 g/mol

BAA5200 Boc-L-HCys(Trt)-OH

N-alpha-t-Butyloxycarbonyl-S-trityl-L-homocysteine

CAS-No. 201419-16-1

Formula C 28 H31NO4 S Mol. weight 477,63 g/mol

BAA5190 Boc-D-HCys(Trt)-OH

N-alpha-t-Butyloxycarbonyl-S-trityl-D-homocysteine

CAS-No. 1301706-43-3

Formula C 28 H31NO4 S Mol. weight 477,63 g/mol

FAA1602 Fmoc-L-HCys(Trt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-trityl-L-homocysteine

CAS-No. 167015-23-8

Formula C 38 H33 NO4 S Mol. weight 599,76 g/mol

FAA3840 Fmoc-L-HCys(Mmt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(4-methoxytrityl)-L-homocysteine

CAS-No. 887644-62-4

Formula C 39 H35NO 5 S Mol. weight 629,76 g/mol

FAA5680 Fmoc-L-HCys(MBzl)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(4-methylbenzyl)-L-homocysteine

CAS-No. 1821768-91-5

Formula C 27H27NO4 S Mol. weight 461,57 g/mol

FAA6120 Fmoc-D-HCys(Trt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-trityl-D-homocysteine

CAS-No. 1007840-62-1

Formula C 38 H33 NO4 S Mol. weight 599,76 g/mol

FAA4830 Fmoc-Nhcys(Trt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-N-[2-(tritylthio)ethyl]-glycine

CAS-No. 882847-27-0

Formula C 38 H33 NO4 S Mol. weight 599,74 g/mol

FAA8865 Fmoc-L-hCys(SIT)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(sec-isoamyl mercaptan)-L-homocysteine

Formula C 24H29 NO4 S 2 Mol. weight 459,62 g/mol

Cyclic Peptides

FAA8870 Fmoc-L-hCys(o Nv)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(2-nitroveratryl)-L-homocysteine

Formula C 28 H28 N2O 8 S Mol. weight 552,60 g/mol

5.3. Cystine Building Blocks

HAA1156 (H-L-Cys-OH)2

L-Cystine

CAS-No. 56-89-3

Formula C 6 H12N2O4 S 2 Mol. weight 240,29 g/mol

HAA1157 H-L-HCystine

L-HomoCystine

CAS-No. 626-72-2

Formula C 8 H16 N2O4 S 2 Mol. weight 268,36 g/mol

HAA9350 (H-L-Sec-OH)2

L-Selenocystine, (H-Sec)2, (H-L-Sec)2

CAS-No. 29621-88-3

Formula C 6 H12N2O4 Se 2 Mol. weight 334,11 g/mol

BAA3680 (Boc-L-Sec)2

N-alpha-t-Butyloxycarbonyl-L-selenocystine

CAS-No. 877754-71-7

Formula C16 H28 N2O 8 Se 2 Mol. weight 534,35 g/mol

BAA5390 (Boc-L-Cys-OH)2

N-alpha,N-alpha‘-di-Boc-L-cystine

CAS-No. 10389-65-8

Formula C16 H28 N2O 8 S 2 Mol. weight 440,52 g/mol

BAA2180 Boc-Cystamine-Suc-OH

4-(2-((2-t-Butyloxycarbonylaminoethyl)disulfanyl) ethylamino)-4-oxobutanoic acid

CAS-No. 946849-79-2

Formula C13 H24N2O 5 S 2 Mol. weight 352,47 g/mol

BNN1360 Di-Boc-Cystamine

N,N‘-Bis-tert-butoxycarbonyl-cystamine

CAS-No. 67385-10-8

Formula C14H28 N2O4 S 2 Mol. weight 352,51 g/mol

ZAA1190 (Z-L-Cys-OH)2

N-alpha-Benzyloxycarbonyl-L-cystine

CAS-No. 6968-11-2

Formula C 22H24N2O 8 S 2 Mol. weight 508,54 g/mol

5.4. Cysteine Protected as Thiazolidine and other Building Blocks

FAA1495 Fmoc-D-Thz-OH

(S)-N-alpha-(9-Fluorenylmethyloxycarbonyl)-thiazolidine-4-carboxylic acid

CAS-No. 198545-89-0

Formula C19 H17NO4 S Mol. weight 355,42 g/mol

Cyclic Peptides

HAA3840 H-L-MeAla(4-Thz)-OH

N-alpha-Methyl-beta-(4-thiazolyl)-L-alanine

CAS-No. 2131118-50-6

Formula C 7H10 N2O 2 S Mol. weight 186,23 g/mol

FAA1427 Fmoc-L-Thz-OH

(R)-N-(9-Fluorenylmethyloxycarbonyl)-thiazolidine-L-4-carboxylic acid

CAS-No. 133054-21-4

Formula C19 H17NO4 S Mol. weight 355,42 g/mol

BAA1135 Boc-L-Thz-OH

(R)-N-t-Butyloxycarbonyl-thiazolidine-4-carboxylic acid

CAS-No. 51077-16-8

Formula C9 H15NO4 S Mol. weight 233,29 g/mol

FAA1437 Fmoc-L-Thz(Me2)-OH

(R)-N-(9-Fluorenylmethyloxycarbonyl)-2,2-dimethyl-thiazolidine-4-carboxylic acid

CAS-No. 873842-06-9

Formula C 21H21NO4 S Mol. weight 383,46 g/mol

BAA1186 Boc-D-Thz-OH

(S)-N-(t-Butyloxycarbonyl)-thiazolidine-4-carboxylic acid

CAS-No. 63091-82-7

Formula C9 H15NO4 S Mol. weight 233,29 g/mol

HAA1132 H-L-Thz-OH

(R)-Thiazolidine-4-carboxylic acid

CAS-No. 34592-47-7

Formula C 4H7NO 2 S Mol. weight 133,16 g/mol

FAA5680 Fmoc-L-HCys(MBzl)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(4-methylbenzyl)-L-homocysteine

CAS-No. 1821768-91-5

Formula C 27H27NO4 S Mol. weight 461,57 g/mol

FAA5370 Fmoc-2-amino-5-(tritylthio)-pentanoic acid (S)

(S)-2-((9-Fluorenylmethyloxycarbonyl)amino)-5-(tritylthio)pentanoic acid

CAS-No. 1417789-17-3

Formula C 39 H35NO4 S Mol. weight 613,76 g/mol

BAA5850 Boc-L-Pen(MBzl)-OH*DCHA

N-alpha-t-Butyloxycarbonyl-S-(4-methylbenzyl)-L-penicillamine dicyclohexylamine

CAS-No. 198474-61-2

Formula C18 H27NO4 S*C12H23 N Mol. weight 353,48*181,32 g/mol

FAA1587 Fmoc-L-Pen(Trt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-trityl-L-penicillamine

CAS-No. 201531-88-6

Formula C 39 H35NO4 S Mol. weight 613,78 g/mol

FAA1675 Fmoc-D-Pen(Trt)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-trityl-D-penicillamine

CAS-No. 201532-01-6

Formula C 39 H35NO4 S Mol. weight 613,78 g/mol

Cyclic Peptides Index

BAA5390 (Boc-L-Cys-OH)2

(Boc-L-Sec)2

Ac-L-Cys-OH

Boc-D-Cys(MBzl)-OH

Boc-D-HCys(MBz)-OH

Boc-L-Cys(Mob)-OH

BAA1860 Boc-L-Cys(Npys)-OH

BAA2250 Boc-L-Cys(Propargyl)-OH*DCHA

BAA6415 Boc-L-Cys(StBu)-OH

BAA1082 Boc-L-Cys(tBu)-OH

BAA1084 Boc-L-Cys(Trt)-OH 5, 73

BAA1083 Boc-L-Cys-OH

BAA5200 Boc-L-HCys(Trt)-OH 83

BAA5850 Boc-L-Pen(MBzl)-OH*DCHA 88

BAA3760 Boc-L-Sec(Mob)-OH 63

BAA4830 Boc-L-Sec(Mob)-OPfp 63

IAD1040 Boc-L-Ser[Fmoc-L-Cys(Trt)]-OH 8 BAA4880 Boc-L-Sez-OH 64

IAD2040 Boc-L-Thr[Fmoc-L-Cys(Trt)]-OH 8

BAA1135 Boc-L-Thz-OH 30, 87

(S)

SAD1107 Fmoc-D-Cys(Acm)-AC TG 23 FAA6230 Fmoc-D-Cys(Acm)-OH

SAD1207 Fmoc-D-Cys(Acm)-Trt TG 23

Fmoc-D-Cys(StBu)-OH 35, 78

SAD1106 Fmoc-D-Cys(Trt)-AC TG 9

FAA1035 Fmoc-D-Cys(Trt)-OH 5, 79

SAD1206 Fmoc-D-Cys(Trt)-Trt TG 9

WAA6118 Fmoc-D-Cys(Trt)-Wang Resin 10

SAD1306 Fmoc-D-Cys(Trt)-Wang TG 9

FAA1470 Fmoc-D-Cys-OH*H2O 74

FAA8260 Fmoc-D-HCys(Acm)-OH 83

FAA6120 Fmoc-D-HCys(Trt)-OH 84

FAA1675 Fmoc-D-Pen(Trt)-OH 6, 88

FAA8710 Fmoc-D-Sec(Mob)-OH 63

FAA8600 Fmoc-D-Sec(Xan)-OH 62

FAA1495 Fmoc-D-Thz-OH 31, 86

PSI1440 Fmoc-Gly-L-Cys[PSI(Dmp,H)pro]-OH 20

PYV1170 Fmoc-Gly-NHN=Pyv Resin 53

FAA8445 Fmoc-Hcan(Boc)-OH (S) 51

FAA9375 Fmoc-L-3-(2-cyano-3-pyridyl)-alanine 59

FAA9370 Fmoc-L-3-(2-cyano-4-pyridyl)-alanine 59

PSI1450 Fmoc-L-Ala-L-Cys[PSI(Dmp,H)pro]-OH 20 PYV1100 Fmoc-L-Ala-NHN=Pyv Resin 52

PYV1110 Fmoc-L-Arg(Pbf)-NHN=Pyv Resin

PYV1120 Fmoc-L-Asn(Trt)-NHN=Pyv Resin

PYV1130 Fmoc-L-Asp(OtBu)-NHN=Pyv Resin

PSI1470 Fmoc-L-Asp(tBu)-L-Cys[PSI(Dmp,H)pro]-OH

FAA8460 Fmoc-L-cis-Hyp(NHBoc)-OH

FAA9395 Fmoc-L-Cys(2,6-DCP)-OH

FAA9250 Fmoc-L-Cys(2-Boc-aminoethyl)-OH

FAA5150 Fmoc-L-Cys(Aapam)-OH

FAA4751 Fmoc-L-Cys(Ac-OtBu)-OH*DCHA

SAL1107 Fmoc-L-Cys(Acm)-AC TG

FAA1506 Fmoc-L-Cys(Acm)-OH

TCP1110

FAA3720 Fmoc-L-Cys(Biotin)-OH

FAA6940

FAA3190 Fmoc-L-Cys(Dpm)-OH

FAA4760 Fmoc-L-Cys(EtCO-OtBu)-OH

FAA7945 Fmoc-L-Cys(MDNPE)-OH

RAA2620 Fmoc-L-Cys(Mmt resin)-NH2

FAA1030 Fmoc-L-Cys(Mmt)-OH

FAA1715 Fmoc-L-Cys(Mob)-OH

FAA4155 Fmoc-L-Cys(Msbh)-OH

FAA8420 Fmoc-L-Cys(NDBF)-OH

FAA5890 Fmoc-L-Cys(Octyl)-OH 80

FAA3970 Fmoc-L-Cys(oNv)-OH 32, 82

FAA1950 Fmoc-L-Cys(Palm)-OH 80

FAA8770 Fmoc-L-Cys(palmityl)-OH

FAA4820

FAA6910 Fmoc-L-Cys(Phacm)-OH

FAA3370 Fmoc-L-Cys(PrCO-OtBu)-OH

FAA3810

FAA3180

FAA8495 Fmoc-L-Cys(SIT)-OH

tBu)-AC TG

Fmoc-L-Cys(SS-tBu)-Trt TG

Fmoc-L-Cys(Trt)-NHN=Pyv Resin

Fmoc-L-Dab(2,6-DCP)-OH

Fmoc-L-Dab(Boc-Cys(Trt))-OH 5, 60

FAA9330 Fmoc-L-Dab(Boc-Thz)-OH 31, 60

FAA9380 Fmoc-L-Dap(2-CINA)-OH 59

FAA9385 Fmoc-L-Dap(6-CNA)-OH 59

PYV1160 Fmoc-L-Gln(Trt)-NHN=Pyv Resin 53

PSI1490 Fmoc-L-Glu(tBu)-L-Cys[PSI(Dmp,H)pro]-OH 20

PYV1150 Fmoc-L-Glu(tBu)-NHN=Pyv Resin 52

FAA8255 Fmoc-L-HCys(Acm)-OH 83

FAA5680 Fmoc-L-HCys(MBzl)-OH 84, 88

FAA3840 Fmoc-L-HCys(Mmt)-OH 84

FAA8870 Fmoc-L-hCys(oNv)-OH 32, 85

FAA8865 Fmoc-L-hCys(SIT)-OH 39, 84

FAA1602 Fmoc-L-HCys(Trt)-OH 84

PYV1180 Fmoc-L-His(Trt)-NHN=Pyv Resin 53

PYV1190 Fmoc-L-Ile-NHN=Pyv Resin 53

PSI1510 Fmoc-L-Leu-L-Cys[PSI(Dmp,H)pro]-OH 20

PYV1200 Fmoc-L-Leu-NHN=Pyv Resin 53

Cyclic Peptides

FAA9340 Fmoc-L-Lys(4-Thz, Boc)-OH

FAA9335 Fmoc-L-Lys(5-STrt, Boc)-OH

PSI1520 Fmoc-L-Lys(Boc)-L-Cys[PSI(Dmp,H)pro]-OH

PYV1210 Fmoc-L-Lys(Boc)-NHN=Pyv Resin

FAA9315 Fmoc-L-Lys(Boc-Cys(Trt))-OH

FAA9320 Fmoc-L-Lys(Boc-Thz)-OH

FAA6720 Fmoc-L-MeCys(Acm)-OH

FAA3340 Fmoc-L-MeCys(S-tBu)-OH

FAA3570 Fmoc-L-MeCys(Trt)-OH

PYV1220 Fmoc-L-Met-NHN=Pyv Resin

PYV1230

tBu)-NHN=Pyv Resin

PYV1260

FAA8455

trans-Hyp(NHBoc)-OH

PYV1270 Fmoc-L-Trp(Boc)-NHN=Pyv Resin

PYV1280 Fmoc-L-Tyr(tBu)-NHN=Pyv Resin

PSI1570 Fmoc-L-Val-L-Cys[PSI(Dmp,H)pro]-OH

PYV1290 Fmoc-L-Val-NHN=Pyv Resin

FAA4830 Fmoc-Nhcys(Trt)-OH 84

PYV1000 Fmoc-NHN=Pyv Resin 51

HAA6110 H-D-Cys(Bzl)-OH

HAA3500 H-D-Cys(Mmt)-OH

RAA1060 H-D-Cys(Trt)-2CT Resin 8

H-D-Cys(Trt)-OH

HAA2100 H-D-Cys(Trt)-OtBu*HCl 7, 70

HAA1017 H-D-Cys-OH*HCl*H2O

HAA1078

RAA1055

Resin

HAA3510 H-L-Cys(Npys)-OH*HCl 39, 72

HAA9320 H-L-Cys(oNv)-OH 32, 69

HAA2350 H-L-Cys(Propargyl)-OH*HCl 68

HAA6140 H-L-Cys(StBu)-OH 35, 69

HAA6150 H-L-Cys(tBu)-OH*HCl 15, 69

RAA1065 H-L-Cys(Trt)-2CT Resin 8 RAA1066 H-L-Cys(Trt)-2CT Resin 8

H-L-Cys(Trt)-NH2 6, 69 HAA6160 H-L-Cys(Trt)-OH 7, 69 HAA1995

oNv)-OH*TFA

Notes

Cyclic Peptides

Code of Conduct

As business activity of Iris Biotech GmbH impacts people’s lives and health, it must be operated in ethical and correct manner and act with integrity and responsibility. To ensure high ethical standards and fair business practices, Iris Biotech GmbH applies an integrated policy known as its Code of Conduct.

In 2001 Iris Biotech GmbH was founded just at the beginning of the Biotech movement and the first remarkable breakthrough of biotech pharma products. Although the biotech field is rather young compared to other industries we believe on long-term business, a good partnership between our business partners and Iris Biotech GmbH and a good reputation. It is our duty as well as our responsibility to maintain and to extend this over the next generations – based on the principles of an honourable and prudent tradesman which based upon the concept of honourable entrepreneurship.

This Code of Conduct has been developed following the “Voluntary Guidelines for Manufacturers of Fine Chemical Intermediates and Active Ingredients” issued by AIME (Agrochemical & Intermediates Manufacturers in Europe) and the requirements of some of our business associates.

Iris Biotech GmbH commits to hold this Code of Conduct and to include and apply its principles in the management system and the company policies.

Ethics

Iris Biotech GmbH undertakes business in an ethical manner and acts with integrity. All corruption, extortion and embezzlement are prohibited. We do not pay or accept bribes or participate in other illegal inducements in business or government relationships. We conduct our business in compliance with all applicable anti-trust laws. Employees are encouraged to report concerns or illegal activities in the workplace, without threat of reprisal, intimidation or harassment.

Labour

Iris Biotech GmbH is committed to uphold the human rights of workers and to treat them with dignity and respect. Child labour, workplace harassment, discrimination, and harsh and inhumane treatment are prohibited. Iris Biotech GmbH respects the rights of the employees to associate freely, join or not join labour unions, seek representation and join workers’ councils. Employees are paid and their working timetable is established according to applicable wage and labour laws. Employees are able to communicate openly with management regarding working conditions without threat of reprisal, intimidation or harassment.

General Policies

Contracts and Secrecy Agreements are binding and the confidential information received is only used for intended purposes. Clear management and organizational structures exist to provide efficient normal working and to address problems quickly. Know-how is protected and intellectual property isrespected.

Health and Safety

Iris Biotech GmbH provides a safe and healthy working environment to the employees and protects them from overexposure to chemical and physical hazards. Products are produced, stored and shipped under the guidelines of the relevant chemical and safety legislation. Risks and emergency scenarios are identified and evaluated, and their possible impact is minimized by implementing emergency plans and written procedures. Safety information regarding hazardous materials is available to educate, train and protect workers from hazards. Preventive equipment and facilities maintenance is performed at suitable periods to reduce potential hazards. Employees are regularly trained in health and safety matters and are informed about product properties and risk classification when it is required.

Environment

Iris Biotech GmbH operates in an environmentally responsible and efficient manner, minimizing adverse impacts on the environment. Waste streams are managed to ensure a safe handling, movement, storage, recycling and reuse, before and after being generated. Systems to prevent and mitigate accidental spills and releases to the environment are in place. All required environmental permits and licenses are obtained and their operational and reporting requirements are complied with.

Production and Quality Management

A quality management system following the Good Distribution Practices (GDP rules) of Active Pharmaceutical Ingredients is established covering all the aspects of the worldwide distribution of products. Regular audits are performed to evaluate the efficiency and fulfilling of the quality system. Process controls to provide reproducible product quality are established. There are preventive maintenance procedures to ensure plant reliability and the lowest risk of failure. Staff is trained periodically about GMP and GDP rules. Procedures are established and installations are designed to avoid cross contamination. Batch and analytical records are kept for inspection and audit purposes for suitable periods according guidelines.

Research and Development

Research and development staff education is appropriate to their functional activity and they are trained to develop, optimize and scale-up the processes. Intellectual property is respected and knowhow protected. Development of manufacturing processes reflects the principles of the Green Chemistry according to the American Chemical Society Green Chemistry Institute. Animal testing is not used unless alternatives are not scientifically valid or accepted by regulators. If animal testing is carried out, animals are treated so that pain and stress are minimized.

Cyclic Peptides

Terms

and Conditions of Sales

All orders placed by a buyer are accepted and all contracts are made subject to the terms which shall prevail and be effective notwithstanding any variations or additions contained in any order or other document submitted by the buyer. No modification of these terms shall be binding upon Iris Biotech GmbH unless made in writing by an authorised representative of Iris Biotech GmbH.

Placing of Orders

Every order made by the buyer shall be deemed an offer by the buyer to purchase products from Iris Biotech GmbH and will not be binding on Iris Biotech GmbH until a duly authorised representative of Iris Biotech GmbH has accepted the offer made by the buyer. Iris Biotech GmbH may accept orders from commercial, educational or government organisations, but not from private individuals and Iris Biotech GmbH reserves the right to insist on a written order and/or references from the buyer before proceeding.

There is no minimum order value. At the time of acceptance of an order Iris Biotech GmbH will either arrange prompt despatch from stock or the manufacture/acquisition of material to satisfy the order. In the event of the latter Iris Biotech GmbH will indicate an estimated delivery date. In addition to all its other rights Iris Biotech GmbH reserves the right to refuse the subsequent cancellation of the order if Iris Biotech GmbH expects to deliver theproduct on or prior to the estimated delivery date. Time shall not be of the essence in respect of delivery of the products. If Iris Biotech GmbH is unable to deliver any products by reason of any circumstances beyond its reasonable control („Force Majeure“) then the period for delivery shall be extended by the time lost due to such Force Majeure. Details of Force Majeure will be forwarded by Iris Biotech GmbH to the buyer as soon as reasonably practicable.

Prices, Quotations and Payments

Prices are subject to change. For the avoidance of doubt, the price advised by Iris Biotech GmbH at the time of the buyer placing the order shall supersede any previous price indications. The buyer must contact the local office of Iris Biotech GmbH before ordering if further information is required. Unless otherwise agreed by the buyer and Iris Biotech GmbH, the price shall be for delivery ex-works. In the event that the buyer requires delivery of the products otherwise than ex-works the buyer should contact the local office of Iris Biotech GmbH in order to detail its requirements. Iris Biotech GmbH shall, at its discretion, arrange the buyer‘s delivery requirements including, without limitation, transit insurance, the mode of transit (Iris Biotech GmbH reserves the right to vary the mode of transit if any regulations or other relevant considerations so require) and any special packaging requirements (including cylinders). For the avoidance of doubt all costs of delivery and packaging in accordance with the buyer‘s requests over and above that of delivery in standard packaging ex-works shall be for the buyer‘s account unless otherwise agreed by both parties. Incoterms 2020 shall apply. Any tax, duty or charge imposed by governmental authority or otherwise and any other applicable taxes, duties or charges shall be for the buyer‘s account. Iris Biotech GmbH may, on request and where possible, provide quotations for multiple packs or bulk quantities, and non-listed items. Irrespective of the type of request or means of response all quotations must be accepted by the buyer without condition and in writing before an order will be accepted by Iris Biotech GmbH. Unless agreed in writing on different terms, quotations are valid for 30 days from the date thereof. Payment terms are net 30 days from invoice date unless otherwise agreed in writing. Iris Biotech GmbH reserves the right to request advance payment at its discretion. For overseas transactions the buyer shall pay all the banking charges of Iris Biotech GmbH. The buyer shall not

be entitled to withhold or set-off payment for the products for any reason whatsoever. Government/ Corporate Visa and MasterCard (and other such credit cards) may be accepted on approved accounts for payment of the products. Personal credit cards are not acceptable. Failure to comply with the terms of payment of Iris Biotech GmbH shall constitute default without reminder. In these circumstances Iris Biotech GmbH may (without prejudice to any other of its rights under these terms) charge interest to accrue on a daily basis at the rate of 2% per month from the date upon which payment falls due to the actual date of payment (such interest shall be paid monthly). If the buyer shall fail to fulfil the payment terms in respect of any invoice of Iris Biotech GmbH Iris Biotech GmbH may demand payment of all outstanding balances from the buyer whether due or not and/or cancel all outstanding orders and/or decline to make further deliveries or provision of services except upon receipt of cash or satisfactory securities. Until payment by the buyer in full of the price and any other monies due to Iris Biotech GmbH in respect of all other products or services supplied or agreed to be supplied by Iris Biotech GmbH to the buyer (including but without limitation any costs of delivery) the property in the products shall remain vested in Iris Biotech GmbH.

Shipping, Packaging and Returns

The buyer shall inspect goods immediately on receipt and inform Iris Biotech GmbH of any shortage or damage within five days. Quality problems must be notified within ten days of receipt. Goods must not be returned without prior written authorisation of Iris Biotech GmbH. Iris Biotech GmbH shall at its sole discretion replace the defective products (or parts thereof) free of charge or refund the price (or proportionate price) to buyer. Opened or damaged containers cannot be returned by the buyer without the written prior agreement of Iris Biotech GmbH. In the case of agreed damaged containers which cannot be so returned, the buyer assumes responsibility for the safe disposal of such containers in accordance with all applicable laws.

Product Quality, Specifications and Technical Information

Products are analysed in the Quality Control laboratories of Iris Biotech GmbH’s production partners by methods and procedures which Iris Biotech GmbH considers appropriate. In the event of any dispute concerning reported discrepancies arising from the buyer’s analytical results, determined by the buyer’s own analytical procedures, Iris Biotech GmbH reserves the right to rely on the results of own analytical methods of Iris Biotech GmbH. Certificates of Analysis or Certificates of Conformity are available at the discretion of Iris Biotech GmbH for bulk orders but not normally for prepack orders. Iris Biotech GmbH reserves the right to make a charge for such certification. Specifications may change and reasonable variation from any value listed should not form the basis of a dispute. Any supply by Iris Biotech GmbH of bespoke or custom product for a buyer shall be to a specification agreed by both parties in writing. Technical information, provided orally, in writing, or by electronic means by or on behalf of Iris Biotech GmbH, including any descriptions, references, illustrations or diagrams in any catalogue or brochure, is provided for guidance purposes only and is subject to change.

Safety

All chemicals should be handled only by competent, suitably trained persons, familiar with laboratory procedures and potential chemical hazards. The burden of safe use of the products of Iris Biotech GmbH vests in the buyer. The buyer assumes all responsibility for warning his employees, and any persons who might reasonably be expected to come into contact with the products, of all risks to person and property in any way connected with the products and for instructing them in their safe handling and use. The buyer also assumes the responsibility for the safe disposal of all products in accordance with all applicable laws.

Cyclic Peptides

Uses, Warranties and Liabilities

All products of Iris Biotech GmbH are intended for laboratory research purposes and unless otherwise stated on product labels, in the catalogue and product information sheet of Iris Biotech GmbH or in other literature furnished to the buyer, are not to be used for any other purposes, including but not limited to use as or as components in drugs for human or animal use, medical devices, cosmetics, food additives, household chemicals, agricultural or horticultural products or pesticides. Iris Biotech GmbH offers no warranty regarding the fitness of any product for a particular purpose and shall not be responsible for any loss or damage whatsoever arising there from. No warranty or representation is given by Iris Biotech GmbH that the products do not infringe any letters patent, trademarks, registered designs or other industrial rights. The buyer further warrants to Iris Biotech GmbH that any use of the products in the United States of America shall not result in the products becoming adulterated or misbranded within the meaning of the Federal Food, Drug and Cosmetic Act (or such equivalent legislation in force in the buyer‘s jurisdiction) and shall not be materials which may not, under sections 404, 505 or 512 of the Act, be introduced into interstate commerce. The buyer acknowledges that, since the products of Iris Biotech GmbH are intended for research purposes, they may not be on the Toxic Substances Control Act 1976 („TSCA“) inventory. The buyer warrants that it shall ensure that the products are approved for use under the TSCA (or such other equivalent legislation in force in the buyer‘s jurisdiction), if applicable. The buyer shall be responsible for complying with any legislation or regulations governing the use of the products and their importation into the country of destination (for the avoidance of doubt to include, without limitation, the TSCA and all its amendments, all EINECS, ELINCS and NONS regulations). If any licence or consent of any government or other authority shall be required for the acquisition, carriage or use of the products by the buyer the buyer shall obtain the same at its own expense and if necessary produce evidence of the same to Iris Biotech GmbH on demand. Failure to do so shall not entitle the buyer to withhold or delay payment. Any additional expenses or charges incurred by Iris Biotech GmbH resulting from such failure shall be for the buyer‘s account. Save for death or personal injury caused by negligence of Iris Biotech GmbH, sole obligation of Iris Biotech GmbH and buyer‘s exclusive remedy with respect to the products proved to the satisfaction of Iris Biotech GmbH to be defective or products incorrectly supplied shall be to accept the return of said products to Iris Biotech GmbH for refund of the actual purchase price paid by the buyer (or proportionate part thereof), or replacement of the defective product (or part thereof) with alternative product. Iris Biotech GmbH shall have no liability to the buyer under or arising directly or indirectly out of or otherwise in connection with the supply of products by Iris Biotech GmbH to the buyer and/or their re-sale or use by the buyer or for any product, process or services of the buyer which in any way comprises the product in contract tort (including negligence or breach of statutory duty) or otherwise for pure economic loss, loss of profit, business, reputation, depletion of brand, contracts, revenues or anticipated savings or for any special indirect or consequential damage or loss of any nature except as may otherwise be expressly provided for in these terms. All implied warranties, terms and representations in respect of the products (whether implied by statute or otherwise) are excluded to the fullest extent permitted by law. The buyer shall indemnify Iris Biotech GmbH for and against any and all losses, damages and expenses, including legal fees and other costs of defending any action, that Iris Biotech GmbH may sustain or incur as a result of any act or omission by the buyer, its officers, agents or employees, its successors or assignees, its customers or all other third parties, whether direct or indirect, in connection with the use of any product. For the avoidance of doubt and in the event that Iris Biotech GmbH supplies bespoke or custom product to the buyer‘s design or specification, this indemnity shall extend to include any claim by a third party that the manufacture of the product for the buyer or the use of the product by the buyer infringes the intellectual property rights of any third party.

General

Iris Biotech GmbH shall be entitled to assign or sub-contract all or any of its rights and obligations hereunder. The buyer shall not be entitled to assign, transfer, sub-contract or otherwise delegate any of its rights or obligations hereunder. Any delay or forbearance by Iris Biotech GmbH in exercising any right or remedy under these terms shall not constitute a waiver of such right or remedy. If any provision of these terms is held by any competent authority to be invalid or unenforceable in whole or in part the validity of the other provisions of these terms and the remainder of the provision in question shall not be affected. These terms shall be governed by German Law and the German Courts shall have exclusive jurisdiction for the hearing of any dispute between the parties save in relation to enforcement where the jurisdiction of the German Courts shall be non-exclusive.

Get in Contact Distribution Partners

Iris Biotech GmbH

Adalbert-Zoellner-Str. 1 95615 Marktredwitz Germany

+49 (0) 9231 97121-0

+49 (0) 9231 97121-99 info@iris-biotech.de www.iris-biotech.de

The list contains the current distributors of Iris Biotech in different regions of the world. The latest list of distribution partners and contact details is available at: arrow-up-right-from-square www.iris-biotech.de/distribution-partner

China: Chengdu Yoo Technology Co., Ltd.

Japan:

BizCom Japan, Inc.

Shigematsu & Co., Ltd

Cosmo Bio Co., Ltd.

USA & Canada: Peptide Solutions LLC

India, Bangladesh, Oman, Sri Lanka, United Arab Emirates: Sumit Biosciences Pvt Ltd.